Secretion and trafficking molecules

ABSTRACT

The invention provides human secretion and trafficking molecules (SAT) and polynucleotides which identify and encode SAT. The invetion also provides expression vectors, host cells, antibodies, agonists and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of SAT.

TECHNICAL FIELD

[0001] This invention relates to nucleic acid and amino acid sequences of secretion and trafficking molecules and to the use of these sequences in the diagnosis, treatment, and prevention of vesicle trafficking, transport, neurological, autoimmune/inflammatory, and cell proliferative disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of secretion and trafficking molecules.

BACKGROUND OF THE INVENTION

[0002] Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into functionally distinct, membrane-bound compartments. The membranes maintain the essential differences between the cytosol, the extracellular environment, and the lumenal space of each intracellular organelle. Eukaryotic proteins including integral membrane proteins, secreted proteins, and proteins destined for the lumen of organelles are synthesized within the endoplasmic reticulum (ER), delivered to the Golgi complex for post-translational processing and sorting, and then transported to specific intracellular and extracellular destinations. Material is internalized from the extracellular environment by endocytosis, a process essential for transmission of neuronal, metabolic, and proliferative signals; uptake of many essential nutrients; and defense against invading organisms. This intracellular and extracellular movement of protein molecules is termed vesicle trafficking. Trafficking is accomplished by the packaging of protein molecules into specialized vesicles which bud from the donor organelle membrane and fuse to the target membrane (Rothman, J. E and Wieland, F. T. (1996) Science 272:227-234).

[0003] The transport of proteins across the ER membrane involves a process that is similar in bacteria, yeast, and mammals (Gorlich, D. et al. (1992) Cell 71: 489-503). In mammalian systems, transport is initiated by the action of a cytoplasmic signal recognition particle (SRP) which recognizes a signal sequence on a growing, nascent polypeptide and binds the polypeptide and its ribosome complex to the ER membrane through an SRP receptor located on the ER membrane. The signal peptide is cleaved and the ribosome complex, together with the attached polypeptide, becomes membrane bound. The polypeptide is subsequently translocated across the ER membrane and into a vesicle (Blobel, G. and B. Dobberstein (1975) J. Cell Biol. 67:852-862).

[0004] Proteins implicated in the translocation of polypeptides across the ER membrane in yeast include SEC61p, SEC62p, and SEC63p. Mutations in the genes encoding these proteins lead to defects in the translocation process. SEC61 may be of particular importance since certain mutations in the gene for this protein inhibit the translocation of many proteins (Gorlich, supra).

[0005] Mammalian homologs of yeast SEC61 (mSEC61) have been identified in dog and rat (Gorlich, supra). Mammalian SEC61 is also structurally similar to SECYp, the bacterial cytoplasmic membrane translocation protein. mSEC61 is found in tight association with membrane-bound ribosomes. This association is induced by membrane-targeting of nascent polypeptide chains and is weakened by dissociation of the ribosomes into their constituent subunits. mSEC61 is postulated to be a component of a putative protein-conducting channel, located in the ER membrane, to which nascent polypeptides are transferred following the completion of translation by ribosomes (Gorlich, supra).

[0006] Several steps in the transit of material along the secretory and endocytic pathways require the formation of transport vesicles. Specifically, vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane (PM), and tubular extensions of the endosomes. Vesicle formation occurs when a region of membrane buds off from the donor organelle. The membrane-bound vesicle contains proteins to be transported and is surrounded by a proteinaceous coat, the components of which are recruited from the cytosol. The initial budding and coating processes are controlled by a cytosolic ras-like GTP-binding protein, ADP-ribosylating factor (Arf), and adapter proteins (AP). Cytosolic GTP-bound Arf is also incorporated into the vesicle as it forms. Different isoforms of both Arf and AP are involved at different sites of budding. For example, Arfs 1, 3, and 5 are required for Golgi budding, Arf4 for endosomal budding, and Arf6 for plasma membrane budding. Two different classes of coat protein have also been identified. Clathrin coats form on vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi (Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12:575-625).

[0007] In clathrin-based vesicle formation, APs bring vesicle cargo and coat proteins together at the surface of the budding membrane. APs are heterotetrameric complexes composed of two large chains: one chain comprised of an α, γ, δ, or ε chain with β chain, a medium chain (μ), and a small chain (σ). Clathrin binds to APs via the carboxy-terminal appendage domain of the β-adaptin subunit (Le Bourgne, R. and Hoflack, B. (1998) Curr. Opin. Cell. Biol. 10:499-503). AP-1 functions in protein sorting from the TGN and endosomes to compartments of the endosomal/lysosomal system. AP-2 functions in clathrin-mediated endocytosis at the plasma membrane, while AP-3 is associated with endosomes and/or the TGN and recruits integral membrane proteins for transport to lysosomes and lysosome-related organelles. The recently isolated AP-4 complex localizes to the TGN or a neighboring compartment and may play a role in sorting events thought to take place in post-Golgi compartments (Dell'Angelica, E. C. et al. (1999) J. Biol. Chem. 274:7278-7285). Cytosolic GTP-bound Arf is also incorporated into the vesicle as it forms. Another GTP-binding protein, dynamin, forms a ring complex around the neck of the forming vesicle and provides the mechanochemical force required to release the vesicle from the donor membrane. The coated vesicle complex is then transported through the cytosol. During the transport process, Arf-bound GTP is hydrolyzed to GDP and the coat dissociates from the transport vesicle (West, M. A. et al. (1997) J. Cell Biol. 138:1239-1254).

[0008] Coatomer (COP) coats, a second class of coat proteins, form on vesicles derived from the ER and Golgi. COP coats can further be classified as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra). The COP coat consists of two major components, a GTP-binding protein (Arf or Sar) and coat protomer (coatomer). Coatomer is an equimolar complex of seven proteins, termed α-, β-, β′-, γ-, Δ-, ε- and Z-COP. The coatomer complex binds to dilysine motifs contained on the cytoplasmic tails of integral membrane proteins. These include the dilysine-containing retrieval motif of membrane proteins of the ER and dibasic/diphenylamine motifs of members of the p24 family. The p24 family of type I membrane proteins represents the major membrane proteins of COPI vesicles. (Harter, C. and Wieland, F. T. (1998) Proc. Natl. Acad. Sci. USA 95:11649-11654.)

[0009] Vesicles can undergo homotypic,fusing with a same type vesicle, or heterotypic, fusing with a different type vesicle, fusion. Molecules required for appropriate targeting and fusion of vesicles include proteins in the vesicle membrane, the target membrane, and proteins recruited from the cytosol. During budding of the vesicle from the donor compartment, an integral membrane protein, VAMP (vesicle-associated membrane protein) is incorporated into the vesicle. Soon after the vesicle uncoats, a cytosolic prenylated GTP-binding protein, Rab, is inserted into the vesicle membrane. The amino acid sequence of Rab proteins reveals conserved GTP-binding domains characteristic of Ras superfamily members. In the vesicle membrane, GTP-bound Rab interacts with VAMP. Once the vesicle reaches the target membrane, a GTPase activating protein (GAP) in the target membrane converts the Rab protein to the GDP-bound form. A cytosolic protein, guanine-nucleotide dissociation inhibitor (GDI) then removes GDP-bound Rab from the vesicle membrane. Several Rab isoforms have been identified and appear to associate with specific compartments within the cell. For example, Rabs 4, 5, and 11 are associated with the early endosome, whereas Rabs 7 and 9 associate with the late endosome. These differences may provide selectivity in the association between vesicles and their target membranes. (Novick, P., and Zerial, M. (1997) Cur. Opin. Cell Biol. 9:496-504.)

[0010] Docking of the transport vesicle with the target membrane involves the formation of a complex between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNAREs, and certain other membrane and cytosolic proteins. Many of these other proteins have been identified although their exact functions in the docking complex remain uncertain (Tellam, J. T. et al. (1995) J. Biol. Chem. 270:5857-5863; Hata, Y. and Sudhof, T. C. (1995) J. Biol. Chem. 270:13022-13028). N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein (α-SNAP and β-SNAP) are two such proteins that are conserved from yeast to man and function in most intracellular membrane fusion reactions. Sec1 represents a family of yeast proteins that function at many different stages in the secretory pathway including membrane fusion. Recently, mammalian homologs of Sec1, called Munc-18 proteins, have been identified (Katagiri, H. et al. (1995) J. Biol. Chem. 270:4963-4966; Hata et al. supra).

[0011] The SNARE complex involves three SNARE molecules, one in the vesicular membrane and two in the target membrane. Together they form a rod-shaped complex of four α-helical coiled-coils. The membrane anchoring domains of all three SNAREs project from one end of the rod. This complex is similar to the rod-like structures formed by fusion proteins characteristic of the enveloped viruses, such as myxovirus, influenza, filovirus (Ebola), and the HIV and SIV retroviruses (Skehel, J. J., and Wiley, D. C. (1998) Cell 95:871-874). It has been proposed that the SNARE complex is sufficient for membrane fusion, suggesting that the proteins which associate with the complex provide regulation over the fusion event (Weber, T. et al. (1998) Cell 92:759-772). For example, in neurons, which exhibit regulated exocytosis, docked vesicles do not fuse with the presynaptic membrane until depolarization, which leads to an influx of calcium (Bennett, M. K., and Scheller, R. H. (1994) Annu. Rev. Biochem. 63:63-100). Synaptotagmin, an integral membrane protein in the synaptic vesicle, associates with the t-SNARE syntaxin in the docking complex. Synaptotagmin binds calcium in a complex with negatively charged phospholipids, which allows the cytosolic SNAP protein to displace synaptotagmin from syntaxin and fusion to occur. Thus, synaptotagmin is a negative regulator of fusion in the neuron. (Littleton, J. T. et al. (1993) Cell 74:1125-1134.)

[0012] The most abundant membrane protein of synaptic vesicles appears to be the glycoprotein synaptophysin, a 38 kDa protein with four transmembrane domains and two intravesicular loops. Synaptophysin monomers associate into homopolymers which form channels in the synaptic vesicle membrane. Synaptophysin's calcium-binding ability, tyrosine phosphorylation, and widespread distribution in neural tissues suggest a potential role in neurosecretion (Bennett, supra.).

[0013] The transport of proteins into and out of vesicles relies on interactions between cell membranes and a supporting membrane cytoskeleton consisting of spectrin and other proteins. A large family of related proteins called ankyrins participate in the transport process by binding to the membrane skeleton protein spectrin and to a protein in the cell membrane called band 3, a component of an anion channel in the cell membrane. Ankyrins therefore function as a critical link between the cytoskeleton and the cell membrane.

[0014] Originally found in association with erythroid cells, ankyrins are also found in other tissues as well (Birkenmeier, C. S. et al. (1993) J. Biol. Chem. 268:9533-9540). Ankyrins are large proteins (˜1800 amino acids) containing an N-terminal, 89 kDa domain that binds the cell membrane proteins band 3 and tubulin, a central 62 kDa domain that binds the cytoskeletal proteins spectrin and vimentin, and a C-terminal, 55 kDa regulatory domain that functions as a modifier of the binding activities of the other two domains. Individual genes for ankyrin are able to produce multiple ankyrin isoforms by various insertions and deletions. These isoforms are of nearly identical size but may have different functions. In addition, smaller transcripts are produced which are missing large regions of the coding sequences from the N-terminal (band 3 binding), and central (spectrin binding) domains. The existence of such a large family of ankyrin proteins and the observation that more than one type of ankyrin may be expressed in the same cell type suggests that ankyrins may have more specialized functions than simply binding the membrane skeleton to the plasma membrane (Birkenmeier, supra).

[0015] In humans, two isoforms of ankyrin are expressed, alternatively, in developing erythroids and mature erythroids, respectively (Lambert, S. et. al. (1990) Proc. Natl. Acad. Sci. USA 87:1730-1734). A deficiency in erythroid spectrin and ankyrin has been associated with the hemolytic anemia, hereditary spherocytosis (Coetzer, T. L. et al. (1988) New Engl. J. Med. 318:230-234).

[0016] Correct trafficking of proteins is of particular importance for the proper function of epithelial cells, which are polarized into distinct apical and basolateral domains containing different cell membrane components such as lipids and membrane-associated proteins. Certain proteins are flexible and may be sorted to the basolateral or apical side depending upon cell type or growth conditions. For example, the kidney anion exchanger (kAE1) can be retargeted from the apical to the basolateral domain if cells are cultured at higher density. The protein kanadaptin was isolated as a protein which binds to the cytoplasmic domain of kAE1. It also colocalizes with kAE1 in vesicles, but not in the membrane, suggesting that kanadaptin's function is to guide kAE1-containing vesicles to the basolateral target membrane (Chen, J. et al. (1998) J. Biol. Chem. 273:1038-1043).

[0017] Vesicle trafficking is crucial in the process of neurotransmission. Synaptic vesicles carry neurotransmitter molecules from the cytoplasm of a neuron to the synapse. Rab3's are a family of GTP-binding proteins located on synaptic vesicles. The RIM family of proteins are thought to be effectors for Rab3's (Wang, Y. et al. (2000) J. Biol. Chem. 275:20033-20044). Rabphilin-3 is a synaptic vesicle protein. Granuphilins are proteins with homology to rabphilins, and may have a unique role in exocytosis (Wang, J. et al. (1999) J. Biol. Chem. 274:28542-28548).

[0018] The etiology of numerous human diseases and disorders can be attributed to defects in the trafficking of proteins to organelles or the cell surface. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR), glucose-galactose malabsorption syndrome (Na⁺/glucose cotransporter), hypercholesterolemia (low-density lipoprotein (LDL) receptor), and forms of diabetes mellitus (insulin receptor). Abnormal hormonal secretion is linked to disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone; ACTH).

[0019] Cancer cells secrete excessive amounts of hormones or other biologically active peptides. Disorders related to excessive secretion of biologically active peptides by tumor cells include: fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and secretion of biologically active peptides (peptides not expected from a tumor) includes ACTH and vasopressin in lung and pancreatic cancers; parathyroid hormone in lung and bladder cancers; calcitonin in lung and breast cancers; and thyroid-stimulating hormone in medullary thyroid carcinoma.

[0020] Various human pathogens alter host cell protein trafficking pathways to their own advantage. For example, the HIV protein Nef down-regulates cell surface expression of CD4 molecules by accelerating their endocytosis through clathrin coated pits. This function of Nef is important for the spread of HIV from the infected cell (Harris, M. (1999) Curr. Biol. 9:R449-R461). A recently identified human protein, Nef-associated factor 1 (Naf1), a protein with four extended coiled-coil domains, has been found to associate with Nef. Overexpression of Naf1 increased cell surface expression of CD4, an effect which could be suppressed by Nef (Fukushi, M. et al. (1999) FEBS Lett. 442:83-88).

[0021] The discovery of new secretion and trafficking molecules and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of vesicle trafficking, transport, neurological autoimmune/inflammatory, and cell proliferative disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of secretion and trafficking molecules.

SUMMARY OF THE INVENTION

[0022] The invention features purified polypeptides, secretion and trafficking molecules, referred to collectively as “SAT” and individually as “SAT-1,” “SAT-2,” “SAT-3,” “SAT-4,” “SAT-5,” “SAT-6,” “SAT-7, ” “SAT-8,” and “SAT-9.” In one aspect, the invention provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. In one alternative, the invention provides an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:1-9.

[0023] The invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO:1-9. In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO:10-18.

[0024] Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. In one alternative, the invention provides a cell transformed with the recombinant polynucleotide. In another alternative, the invention provides a transgenic organism comprising the recombinant polynucleotide.

[0025] The invention also provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.

[0026] Additionally, the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.

[0027] The invention further provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides.

[0028] Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof. In one alternative, the probe comprises at least 60 contiguous nucleotides.

[0029] The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d). The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.

[0030] The invention further provides a composition comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and a pharmaceutically acceptable excipient. In one embodiment, the composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional SAT, comprising administering to a patient in need of such treatment the composition.

[0031] The invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample. In one alternative, the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional SAT, comprising administering to a patient in need of such treatment the composition.

[0032] Additionally, the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. In another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional SAT, comprising administering to a patient in need of such treatment the composition.

[0033] The invention further provides a method of screening for a compound that specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.

[0034] The invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9. The method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.

[0035] The invention further provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO:10-18, the method comprising a) exposing a sample comprising the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide.

[0036] The invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv). Alternatively, the target polynucleotide comprises a fragment of a polynucleotide sequence selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.

BRIEF DESCRIPTION OF THE TABLES

[0037] Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the present invention.

[0038] Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog for polypeptides of the invention. The probability score for the match between each polypeptide and its GenBank homolog is also shown.

[0039] Table 3 shows structural features of polypeptide sequences of the invention, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.

[0040] Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide sequences of the invention, along with selected fragments of the polynucleotide sequences.

[0041] Table 5 shows the representative cDNA library for polynucleotides of the invention.

[0042] Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.

[0043] Table 7 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.

DESCRIPTION OF THE INVENTION

[0044] Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0045] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0047] Definitions

[0048] “SAT” refers to the amino acid sequences of substantially purified SAT obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

[0049] The term “agonist” refers to a molecule which intensifies or mimics the biological activity of SAT. Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SAT either by directly interacting with SAT or by acting on components of the biological pathway in which SAT participates.

[0050] An “allelic variant” is an alternative form of the gene encoding SAT. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

[0051] “Altered” nucleic acid sequences encoding SAT include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as SAT or a polypeptide with at least one functional characteristic of SAT. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding SAT, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding SAT. The encoded protein may also be “altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent SAT. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of SAT is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, and positively charged amino acids may include lysine and arginine. Amino acids with uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine. Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.

[0052] The terms “amino acid” and “amino acid sequence” refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0053] “Amplification” relates to the production of additional copies of a nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.

[0054] The term “antagonist” refers to a molecule which inhibits or attenuates the biological activity of SAT. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of SAT either by directly interacting with SAT or by acting on components of the biological pathway in which SAT participates.

[0055] The term “antibody” refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which are capable of binding an epitopic determinant. Antibodies that bind SAT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.

[0056] The term “antigenic determinant” refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

[0057] The term “antisense” refers to any composition capable of base-pairing with the “sense” (coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may be produced by any method including chemical synthesis or transcription. Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occurring nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation. The designation “negative” or “minus” can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.

[0058] The term “biologically active” refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. likewise, “immunologically active” or “immunogenic” refers to the capability of the natural, recombinant, or synthetic SAT, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

[0059] “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

[0060] A “composition comprising a given polynucleotide sequence” and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding SAT or fragments of SAT may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk salmon sperm DNA, etc.).

[0061] “Consensus sequence” refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.) in the 5′ and/or the 3′ direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison Wis.) or Phrap (University of Washington, Seattle Wash.). Some sequences have been both extended and assembled to produce the consensus sequence.

[0062] “Conservative amino acid substitutions” are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions. The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions. Original Residue Conservative Substitution Ala Gly, Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu, Thr

[0063] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

[0064] A “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

[0065] The term “derivative” refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.

[0066] A “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.

[0067] “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.

[0068] A “fragment” is a unique portion of SAT or the polynucleotide encoding SAT which is identical in sequence to but shorter in length than the parent sequence. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues. A fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

[0069] A fragment of SEQ ID NO:10-18 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO:10-18, for example, as distinct from any other sequence in the genome from which the fragment was obtained. A fragment of SEQ ID NO:10-18 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:10-18 from related polynucleotide sequences. The precise length of a fragment of SEQ ID NO:10-18 and the region of SEQ ID NO:10-18 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

[0070] A fragment of SEQ ID NO:1-9 is encoded by a fragment of SEQ ID NO:10-18. A fragment of SEQ ID NO:1-9 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO:1-9. For example, a fragment of SEQ ID NO:1-9 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:1-9. The precise length of a fragment of SEQ ID NO:1-9 and the region of SEQ ID NO:1-9 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.

[0071] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0072] “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.

[0073] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

[0074] Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D. G. et al. (1992) CABIOS 8:189-191. For pairwise alignments of polynucleotide sequences, the default parameters are set as follows: Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The “weighted” residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the “percent similarity” between aligned polynucleotide sequences.

[0075] Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at http://www.ncbi.nlm.nih.gov/gorf/b12.html. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at default parameters. Such default parameters may be, for example:

[0076] Matrix: BLOSUM62

[0077] Reward for match: 1

[0078] Penalty for mismatch: −2

[0079] Open Gap: 5 and Extension Gap: 2 penalties

[0080] Gap x drop-off: 50

[0081] Expect: 10

[0082] Word Size: 11

[0083] Filter: on

[0084] Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0085] Nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.

[0086] The phrases “percent identity” and “% identity,” as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide.

[0087] Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above). For pairwise alignments of polypeptide sequences using CLUSTAL V, the default parameters are set as follows: Ktuple=1, gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the “percent similarity” between aligned polypeptide sequence pairs.

[0088] Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypeptide sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set at default parameters. Such default parameters may be, for example:

[0089] Matrix: BLOSUM62

[0090] Open Gap: 11 and Extension Gap: 1 penalties

[0091] Gap x drop-off: 50

[0092] Expect: 10

[0093] Word Size: 3

[0094] Filter: on

[0095] Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0096] “Human artificial chromosomes” (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain all of the elements required for chromosome replication, segregation and maintenance.

[0097] The term “humanized antibody” refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.

[0098] “Hybridization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the “washing” step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched. Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68° C. in the presence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/ml sheared, denatured salmon sperm DNA.

[0099] Generally, stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out. Such wash temperatures are typically selected to be about 5° C. to 20° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength and pH. The T_(m) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. An equation for calculating T_(m) and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.; specifically see volume 2, chapter 9.

[0100] High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68° C. in the presence of about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSC concentration may be varied from about 0.1 to 2×SSC, with SDS being present at about 0.1%. Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA:DNA hybridizations. Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.

[0101] The term “hybridization complex” refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (e.g., C₀t or R₀t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).

[0102] The words “insertion” and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.

[0103] “Immune response” can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemolines, and other signaling molecules, which may affect cellular and systemic defense systems.

[0104] An “immunogenic fragment” is a polypeptide or oligopeptide fragment of SAT which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal. The term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of SAT which is useful in any of the antibody production methods disclosed herein or known in the art.

[0105] The term “microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, or other chemical compounds on a substrate.

[0106] The terms “element” and “array element” refer to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.

[0107] The term “modulate” refers to a change in the activity of SAT. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of SAT.

[0108] The phrases “nucleic acid” and “nucleic acid sequence” refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.

[0109] “Operably linked” refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0110] “Peptide nucleic acid” (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.

[0111] “Post-translational modification” of an SAT may involve lipidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur synthetically or biochemically. Biochemical modifications will vary by cell type depending on the enzymatic milieu of SAT.

[0112] “Probe” refers to nucleic acid sequences encoding SAT, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes. “Primers” are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).

[0113] Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. in order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.

[0114] Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, Cold Spring Harbor Press, Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols in Molecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New York N.Y.; Innis, M. et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, San Diego Calif. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge Mass.).

[0115] Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University of Texas South West Medical Center, Dallas Tex.) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge Mass.) allows the user to input a “mispriming library,” in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.) The PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments. The oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.

[0116] A “recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra. The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid. Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.

[0117] Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.

[0118] A “regulatory element” refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability.

[0119] “Reporter molecules” are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.

[0120] An “RNA equivalent,” in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

[0121] The term “sample” is used in its broadest sense. A sample suspected of containing SAT, nucleic acids encoding SAT, or fragments thereof may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.

[0122] The terms “specific binding” and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope “A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

[0123] The term “substantially purified” refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

[0124] A “substitution” refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides, respectively.

[0125] “Substrate” refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.

[0126] A “transcript image” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.

[0127] “Transformation” describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment. The term “transformed cells” includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.

[0128] A “transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. The transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals. The isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.

[0129] A “variant” of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool Version 2.0.9 (May 07, 1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length. A variant may be described as, for example, an “allelic” (as defined above), “splice,” “species,” or “polymorphic” variant A splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides will generally have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass “single nucleotide polymorphisms” (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.

[0130] A “variant” of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool Version 2.0.9 (May 07, 1999) set at default parameters. Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.

[0131] The Invention

[0132] The invention is based on the discovery of new human secretion and trafficking molecules (SAT), the polynucleotides encoding SAT, and the use of these compositions for the diagnosis, treatment, or prevention of vesicle trafficking, transport, neurological, autoimmune/inflammatory, and cell proliferative disorders.

[0133] Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide sequences of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown. Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.

[0134] Table 2 shows sequences with homology to the polypeptides of the invention as identified by BLAST analysis against the GenBank protein (genpept) database. Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention. Column 3 shows the GenBank identification number (Genbank ID NO:) of the nearest GenBank homolog. Column 4 shows the probability score for the match between each polypeptide and its GenBank homolog. Column 5 shows the annotation of the GenBankhomolog along with relevant citations where applicable, all of which are expressly incorporated by reference herein.

[0135] Table 3 shows various structural features of the polypeptides of the invention. Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention. Column 3 shows the number of amino acid residues in each polypeptide. Column 4 shows potential phosphorylation sites, and column 5 shows potential glycosylation sites, as determined by the MOTIFS program of the GCG sequence analysis software package (Genetics Computer Group, Madison Wis.). Column 6 shows amino acid residues comprising signature sequences, domains, and motifs. Column 7 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.

[0136] Together, Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties establish that the claimed polypeptides are secretion and trafficking molecules. For example, SEQ ID NO:2 is 93% identical to mitsugumin29 (GenB ank ID g3077703), a synaptophysin family member, as determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST probability score is 2.9e-136, which indicates the probability of obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:2 also contains a synaptophysin/synaptoporin domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS and PROFILESCAN analyses, and BLAST comparisons to protein signature sequences in the DOMO and PRODOM databases provide further corroborative evidence that SEQ ID NO:2 is a synaptophysin family member. SEQ ID NO:3 is 72% identical to rat apical endosomal glycoprotein (GenBank ID g777776) with a BLAST probability score of 0.0. Data from BLAST analyses against the PRODOM database provide further corroborative evidence that SEQ ID NO:3 is an apical endosomal glycoprotein. SEQ ID NO:8 is 95% identical to Rattus norvegicus synaptotagmin III (GenBank ID g484296) with a BLAST probability score of 0.0. SEQ ID NO:8 also contains a C2 domain as determined by searching for statistically significant matches in the hidden Markov model (HMM)-based PFAM database of conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses provide further corroborative evidence that SEQ ID NO:8 is a C2 domain-containing protein, most likely a member of the synaptotagmin family. SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9 were analyzed and annotated in a similar manner. The algorithms and parameters for the analysis of SEQ ID NO:1-9 are described in Table 7.

[0137] As shown in Table 4, the full length polynucleotide sequences of the present invention were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences. Columns 1 and 2 list the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and the corresponding Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) for each polynucleotide of the invention. Column 3 shows the length of each polynucleotide sequence in basepairs. Column 4 lists fragments of the polynucleotide sequences which are useful for example, in hybridization or amplification technologies that identify SEQ ID NO:10-18 or that distinguish between SEQ ID NO:10-18 and related polynucleotide sequences. Column 5 shows identification numbers corresponding to cDNA sequences, coding sequences (exons) predicted from genomic DNA, and/or sequence assemblages comprised of both cDNA and genomic DNA. These sequences were used to assemble the full length polynucleotide sequences of the invention. Columns 6 and 7 of Table 4 show the nucleotide start (5′) and stop (3′) positions of the cDNA and/or genomic sequences in column 5 relative to their respective full length sequences.

[0138] The identification numbers in Column 5of Table 4 may refer specifically, for example, to Incyte cDNAs along with their corresponding cDNA libraries. For example, 1438701F1 is the identification number of an Incyte cDNA sequence, and PANCNOT02 is the cDNA library from which it is derived. Incyte cDNAs for which cDNA libraries are not indicated were derived from pooled cDNA libraries (e.g., 70767606V1). Alternatively, the identification numbers in column 5 may refer to GenBank cDNAs or ESTs (e.g., g5810426) which contributed to the assembly of the full length polynucleotide sequences. In addition, the identification numbers in column 5 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences including the designation “ENST”). Alternatively, the identification numbers in column 5 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those sequences including the designation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e., those sequences including the designation “NP”). Alternatively, the identification numbers in column 5 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an “exon stitching” algorithm. For example, FL_XXXXXX_N_(1—)N_(2—)YYYYY_N_(3—)N₄ represents a “stitched” sequence in which XXXXXX is the identification number of the cluster of sequences to which the algorithm was applied, and YYYYY is the number of the prediction generated by the algorithm, and N_(1,2,3 . . .) , if present, represent specific exons that may have been manually edited during analysis (See Example V). Alternatively, the identification numbers in column 5 may refer to assemblages of exons brought together by an “exon-stretching” algorithm. For example, FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is the identification number of a “stretched” sequence, with XXXXXX being the Incyte project identification number, gAAAAA being the GenBank identification number of the human genomic sequence to which the “exon-stretching” algorithm was applied, gBBBBB being the GenBank identification number or NCBI RefSeq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V). In instances where a RefSeq sequence was used as a protein homolog for the “exon-stretching” algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) may be used in place of the GenB ank identifier (ie., gBBBBB).

[0139] Alternatively, a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods. The following Table lists examples of component sequence prefixes and corresponding sequence analysis methods associated with the prefixes (see Example IV and Example V). Prefix Type of analysis and/or examples of programs GNN, Exon prediction from genomic sequences using, for example, GFG, GENSCAN (Stanford University, CA, USA) or FGENES ENST (Computer Genomics Group, The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis of genomic sequences. FL Stitched or stretched genomic sequences (see Example V).

[0140] In some cases, Incyte cDNA coverage redundant with the sequence coverage shown in column 5 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.

[0141] Table 5 shows the representative cDNA libraries for those full length polynucleotide sequences which were assembled using Incyte cDNA sequences. The representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotide sequences. The tissues and vectors which were used to construct the cDNA libraries shown in Table 5 are described in Table 6.

[0142] The invention also encompasses SAT variants. A preferred SAT variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the SAT amino acid sequence, and which contains at least one functional or structural characteristic of SAT.

[0143] The invention also encompasses polynucleotides which encode SAT. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:10-18, which encodes SAT. The polynucleotide sequences of SEQ ID NO:10-18, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.

[0144] The invention also encompasses a variant of a polynucleotide sequence encoding SAT. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding SAT. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:10-18 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:10-18. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of SAT.

[0145] It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding SAT, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring SAT, and all such variations are to be considered as being specifically disclosed.

[0146] Although nucleotide sequences which encode SAT and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring SAT under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding SAT or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding SAT and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

[0147] The invention also encompasses production of DNA sequences which encode SAT and SAT derivatives, or fragments thereof, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding SAT or any fragment thereof.

[0148] Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:10-18 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511.) Hybridization conditions, including annealing and wash conditions, are described in “Definitions.”

[0149] Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway N.J.), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg Md.). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is then carried out using either the ABI 373 or 377 DNA sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale Calif.), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art (See, e.g., Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

[0150] The nucleic acid sequences encoding SAT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth Minn.) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68° C. to 72° C.

[0151] When screening for fill length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5′ regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5′ non-transcribed regulatory regions.

[0152] Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.

[0153] In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode SAT may be cloned in recombinant DNA molecules that direct expression of SAT, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express SAT.

[0154] The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter SAT-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.

[0155] The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-319) to alter or improve the biological properties of SAT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through “artificial” breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.

[0156] In another embodiment, sequences encoding SAT may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232.) Alternatively, SAT itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techniques. (See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular Properties, W H Freeman, New York N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995) Science 269:202-204.) Automated synthesis maybe achieved using the ABI 431A peptide synthesizer (Applied Biosystems). Additionally, the amino acid sequence of SAT, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturally occurring polypeptide.

[0157] The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g., Chiez, R. M. and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, supra, pp. 28-53.)

[0158] In order to express a biologically active SAT, the nucleotide sequences encoding SAT or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host. These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5′ and 3′ untranslated regions in the vector and in polynucleotide sequences encoding SAT. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding SAT. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding SAT and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0159] Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding SAT and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., ch. 9,13, and 16.)

[0160] A variety of expression vector/host systems may be utilized to contain and express sequences encoding SAT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Buller, R. M. et al. (1985) Nature 317(6040):813-815; McGregor, D. P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I. M. and N. Somia (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.

[0161] In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding SAT. For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding SAT can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding SAT into the vector's multiple cloning site disrupts the lacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large quantities of SAT are needed, e.g. for the production of antibodies, vectors which direct high level expression of SAT may be used. For example, vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.

[0162] Yeast expression systems may be used for production of SAT. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995, supra; Bitter, G. A. et al. (1987) Methods Enzymol. 153:516-544; and Scorer, C. A. et al. (1994) Bio/Technology 12:181-184.)

[0163] Plant systems may also be used for expression of SAT. Transcription of sequences encoding SAT may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York N.Y., pp. 191-196.)

[0164] In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding SAT may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses SAT in host cells. (See, e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for high-level protein expression.

[0165] Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.)

[0166] For long term production of recombinant proteins in mammalian systems, stable expression of SAT in cell lines is preferred. For example, sequences encoding SAT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.

[0167] Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk⁻ and apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), β glucuronidase and its substrate β-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol. 55:121-131.)

[0168] Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding SAT is inserted within a marker gene sequence, transformed cells containing sequences encoding SAT can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding SAT under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.

[0169] In general, host cells that contain the nucleic acid sequence encoding SAT and that express SAT may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.

[0170] Immunological methods for detecting and measuring the expression of SAT using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on SAT is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn., Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.)

[0171] A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding SAT include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding SAT, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0172] Host cells transformed with nucleotide sequences encoding SAT may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode SAT may be designed to contain signal sequences which direct secretion of SAT through a prokaryotic or eukaryotic cell membrane.

[0173] In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a “prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure the correct modification and processing of the foreign protein.

[0174] In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding SAT may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric SAT protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of SAT activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the SAT encoding sequence and the heterologous protein sequence, so that SAT may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.

[0175] In a further embodiment of the invention, synthesis of radiolabeled SAT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, ³⁵S-methionine.

[0176] SAT of the present invention or fragments thereof may be used to screen for compounds that specifically bind to SAT. At least one and up to a plurality of test compounds may be screened for specific binding to SAT. Examples of test compounds include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

[0177] In one embodiment, the compound thus identified is closely related to the natural ligand of SAT, e.g., a ligand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, e.g., Coligan, J. E. et al. (1991) Current Protocols in Immunology 1(2): Chapter 5.) Similarly, the compound can be closely related to the natural receptor to which SAT binds, or to at least a fragment of the receptor, e.g., the ligand binding site. In either case, the compound can be rationally designed using known techniques. In one embodiment, screening for these compounds involves producing appropriate cells which express SAT, either as a secreted protein or on the cell membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing SAT or cell membrane fractions which contain SAT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either SAT or the compound is analyzed.

[0178] An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may comprise the steps of combining at least one test compound with SAT, either in solution or affixed to a solid support, and detecting the binding of SAT to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support.

[0179] SAT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of SAT. Such compounds may include agonists, antagonists, or partial or inverse agonists. In one embodiment, an assay is performed under conditions permissive for SAT activity, wherein SAT is combined with at least one test compound, and the activity of SAT in the presence of a test compound is compared with the activity of SAT in the absence of the test compound. A change in the activity of SAT in the presence of the test compound is indicative of a compound that modulates the activity of SAT. Alternatively, a test compound is combined with an in vitro or cell-free system comprising SAT under conditions suitable for SAT activity, and the assay is performed. In either of these assays, a test compound which modulates the activity of SAT may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.

[0180] In another embodiment, polynucleotides encoding SAT or their mammalian homologs may be “knocked out” in an animal model system using homologous recombination in embryonic stem (ES) cells. Such techniques are well known in the art and are useful for the generation of animal models of human disease. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture. The ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vector integrates into the corresponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J. D. (1996) Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.

[0181] Polynucleotides encoding SAT may also be manipulated in vitro in ES cells derived from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell lineages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

[0182] Polynucleotides encoding SAT can also be used to create “knockin” humanized animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding SAT is injected into animal ES cells, and the injected sequence integrates into the animal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress SAT, e.g., by secreting SAT in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

[0183] Therapeutics

[0184] Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of SAT and secretion and trafficking molecules. In addition, the expression of SAT is closely associated with brain, spinal cord, lymphatic, and reproductive tissues. Therefore, SAT appears to play a role in vesicle trafficking, transport, neurological, autoimmune/inflammatory, and cell proliferative disorders. In the treatment of disorders associated with increased SAT expression or activity, it is desirable to decrease the expression or activity of SAT. In the treatment of disorders associated with decreased SAT expression or activity, it is desirable to increase the expression or activity of SAT.

[0185] Therefore, in one embodiment, SAT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SAT. Examples of such disorders include, but are not limited to, a vesicle trafficking disorder such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease; gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers; other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS); allergies including hay fever, asthma, and urticaria (hives); autoimmune hemolytic anemia; proliferative glomerulonephritis; inflammatory bowel disease; multiple sclerosis; myasthenia gravis; rheumatoid and osteoarthritis; scleroderma; Chediak-Higashi and Sjogren's syndromes; systemic lupus erythematosus; toxic shock syndrome; traumatic tissue damage; and viral, bacterial, fungal, helminthic, and protozoal infections; a transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy, cerebral neoplasms, prostate cancer; cardiac disorders associated with transport, e.g., angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis; neurological disorders associated with transport, e.g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia; and other disorders associated with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes disease, occipital horn syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; an autoimmune/inflammatory disorder such as such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.

[0186] In another embodiment, a vector capable of expressing SAT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SAT including, but not limited to, those described above.

[0187] In a further embodiment, a composition comprising a substantially purified SAT in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SAT including, but not limited to, those provided above.

[0188] In still another embodiment, an agonist which modulates the activity of SAT may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of SAT including, but not limited to, those listed above.

[0189] In a further embodiment, an antagonist of SAT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SAT. Examples of such disorders include, but are not limited to, those vesicle trafficking, transport, neurological, autoimmune/inflammatory, and cell proliferative disorders described above. In one aspect, an antibody which specifically binds SAT may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express SAT.

[0190] In an additional embodiment, a vector expressing the complement of the polynucleotide encoding SAT may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of SAT including, but not limited to, those described above.

[0191] In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0192] An antagonist of SAT may be produced using methods which are generally known in the art. In particular, purified SAT may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind SAT. Antibodies to SAT may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.

[0193] For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with SAT or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especially preferable.

[0194] It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to SAT have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein. Short stretches of SAT amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.

[0195] Monoclonal antibodies to SAT may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0196] In addition, techniques developed for the production of “chimeric antibodies,” such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See, e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce SAT-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)

[0197] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

[0198] Antibody fragments which contain specific binding sites for SAT may also be generated. For example, such fragments include, but are not limited to, F(ab′)₂ fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science 246:1275-1281.)

[0199] Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between SAT and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering SAT epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).

[0200] Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for SAT. Affinity is expressed as an association constant, K_(a), which is defined as the molar concentration of SAT-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K_(a) determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple SAT epitopes, represents the average affinity, or avidity, of the antibodies for SAT. The K_(a) determined for a preparation of monoclonal antibodies, which are monospecific for a particular SAT epitope, represents a true measure of affinity. High-affinity antibody preparations with K_(a) ranging from about 10⁹ to 10¹² L/mole are preferred for use in immunoassays in which the SAT-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to 10⁷ L/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of SAT, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York N.Y.).

[0201] The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of SAT-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available. (See, e.g., Catty, supra, and Coligan et al. supra.)

[0202] In another embodiment of the invention, the polynucleotides encoding SAT, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding SAT. Such technology is well known in the art, and antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding SAT. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa N.J.)

[0203] In therapeutic use, any gene delivery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J. E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K. J. et al. (1995) 9(13):1288-1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A. D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.) Other gene delivery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J. J. (1995) Br. Med. Bull. 51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.)

[0204] In another embodiment of the invention, polynucleotides encoding SAT may be used for somatic or germline gene therapy. Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familial hypercholesterolemia, and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410; Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proliferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses, such as human immunodeficiency virus (HV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in SAT expression or regulation causes disease, the expression of SAT from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.

[0205] In a further embodiment of the invention, diseases or disorders caused by deficiencies in SAT are treated by constructing mammalian expression vectors encoding SAT and introducing these vectors by mechanical means into SAT-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson (1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).

[0206] Expression vectors that may be effective for the expression of SAT include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). SAT may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V. and Blau, H. M. supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding SAT from a normal individual.

[0207] Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham, F. L. and A. J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.

[0208] In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to SAT expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding SAT under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and (iii) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol. 61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,434 to Rigg (“Method for obtaining retrovirus packaging cell lines producing high transducing efficiency retroviral supernatant”) discloses a method for obtaining retrovirus packaging cell lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of cells (e.g., CD4⁺ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).

[0209] In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding SAT to cells which have one or more genetic abnormalities with respect to the expression of SAT. The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M. E. et al. (1995) Transplantation 27:263-268). Potentially useful adenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano (“Adenovirus vectors for gene therapy”), hereby incorporated by reference. For adenoviral vectors, see also Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and Verma, I. M. and N. Somia (1997) Nature 18:389:239-242, both incorporated by reference herein.

[0210] In another alternative, a herpes-based, gene therapy delivery system is used to deliver polynucleotides encoding SAT to target cells which have one or more genetic abnormalities with respect to the expression of SAT. The use of herpes simplex virus (HSV)-based vectors may be especially valuable for introducing SAT to cells of the central nervous system, for which HSV has a tropism. The construction and packaging of herpes-based vectors are well known to those with ordinary skill in the art. A replication-competent herpes simplex virus (HSV) type 1-based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S. Pat. No. 5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”), which is hereby incorporated by reference. U.S. Pat. No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a cell under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goins, W. F. et al. (1999) J. Virol. 73:519-532 and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of cloned herpesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments of the large herpesvirus genomes, the growth and propagation of herpesvirus, and the infection of cells with herpesvirus are techniques well known to those of ordinary skill in the art.

[0211] In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding SAT to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenomic RNA is generated that normally encodes the viral capsid proteins. This subgenomic RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase). Similarly, inserting the coding sequence for SAT into the alphavirus genome in place of the capsid-coding region results in the production of a large number of SAT-coding RNAs and the synthesis of high levels of SAT in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S. A. et al. (1997) Virology 228:74-83). The wide host range of alphaviruses will allow the introduction of SAT into a variety of cell types. The specific transduction of a subset of cells in a population may require the sorting of cells prior to transduction. The methods of manipulating infectious cDNA clones of alphaviruses, performing alphavirus cDNA and RNA transfections, and performing alphavirus infections, are well known to those with ordinary skill in the art.

[0212] Oligonucleotides derived from the transcription initiation site, e.g., between about positions −10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

[0213] Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding SAT.

[0214] Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0215] Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding SAT. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues.

[0216] RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′ O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

[0217] An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding SAT. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased SAT expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding SAT may be therapeutically useful, and in the treatment of disorders associated with decreased SAT expression or activity, a compound which specifically promotes expression of the polynucleotide encoding SAT may be therapeutically useful.

[0218] At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding SAT is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding SAT are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding SAT. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified oligonucleotides) for antisense activity against a specific polynucleotide sequence (Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. et al. (2000) U.S. Pat. No. 6,022,691).

[0219] Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art. (See, e.g., Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466.)

[0220] Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.

[0221] An additional embodiment of the invention relates to the administration of a composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such compositions may consist of SAT, antibodies to SAT, and mimetics, agonists, antagonists, or inhibitors of SAT.

[0222] The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0223] Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g. larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region of the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No. 5,997,848). Pulmonary delivery has the advantage of administration without needle injection, and obviates the need for potentially toxic penetration enhancers.

[0224] Compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

[0225] Specialized forms of compositions may be prepared for direct intracellular delivery of macromolecules comprising SAT or fragments thereof. For example, liposome preparations. containing a cell-impermeable macromolecule may promote cell fusion and intracellular delivery of the macromolecule. Alternatively, SAT or a fragment thereof may be joined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

[0226] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0227] A therapeutically effective dose refers to that amount of active ingredient, for example SAT or fragments thereof, antibodies of SAT, and agonists, antagonists or inhibitors of SAT, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED₅₀ (the dose therapeutically effective in 50% of the population) or LD₅₀ (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio. Compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

[0228] The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.

[0229] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to a total dose of about 1 gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

[0230] Diagnostics

[0231] In another embodiment, antibodies which specifically bind SAT may be used for the diagnosis of disorders characterized by expression of SAT, or in assays to monitor patients being treated with SAT or agonists, antagonists, or inhibitors of SAT. Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for SAT include methods which utilize the antibody and a label to detect SAT in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

[0232] A variety of protocols for measuring SAT, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of SAT expression. Normal or standard values for SAT expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibodies to SAT under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of SAT expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

[0233] In another embodiment of the invention, the polynucleotides encoding SAT may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of SAT may be correlated with disease. The diagnostic assay may be used to determine absence, presence, and excess expression of SAT, and to monitor regulation of SAT levels during therapeutic intervention.

[0234] In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding SAT or closely related molecules may be used to identify nucleic acid sequences which encode SAT. The specificity of the probe, whether it is made from a highly specific region, e.g., the 5′ regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding SAT, allelic variants, or related sequences.

[0235] Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the SAT encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:10-18 or from genomic sequences including promoters, enhancers, and introns of the SAT gene.

[0236] Means for producing specific hybridization probes for DNAs encoding SAT include the cloning of polynucleotide sequences encoding SAT or SAT derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.

[0237] Polynucleotide sequences encoding SAT may be used for the diagnosis of disorders associated with expression of SAT. Examples of such disorders include, but are not limited to, a vesicle trafficking disorder such as cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabetes insipidus, hyper- and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison's disease; gastrointestinal disorders including ulcerative colitis, gastric and duodenal ulcers; other conditions associated with abnormal vesicle trafficking, including acquired immunodeficiency syndrome (AIDS); allergies including hay fever, asthma, and urticaria (hives); autoimmune hemolytic anemia; proliferative glomerulonephritis; inflammatory bowel disease; multiple sclerosis; myasthenia gravis; rheumatoid and osteoarthritis; scleroderma; Chediak-Higashi and Sjogren's syndromes; systemic lupus erythematosus; toxic shock syndrome; traumatic tissue damage; and viral, bacterial, fungal, helminthic, and protozoal infections; a transport disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, diabetes mellitus, diabetes insipidus, diabetic neuropathy, Duchenne muscular dystrophy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, Parkinson's disease, malignant hyperthermia, multidrug resistance, myasthenia gravis, myotonic dystrophy, catatonia, tardive dyskinesia, dystonias, peripheral neuropathy, cerebral neoplasms, prostate cancer; cardiac disorders associated with transport, e.g., angina, bradyarrythmia, tachyarrythmia, hypertension, Long QT syndrome, myocarditis, cardiomyopathy, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, thyrotoxic myopathy, ethanol myopathy, dermatomyositis, inclusion body myositis, infectious myositis, polymyositis; neurological disorders associated with transport, e.g., Alzheimer's disease, amnesia, bipolar disorder, dementia, depression, epilepsy, Tourette's disorder, paranoid psychoses, and schizophrenia; and other disorders associated with transport, e.g., neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, Cushing's disease, Addison's disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, adrenoleukodystrophy, Zellweger syndrome, Menkes disease, occipital horn syndrome, von Gierke disease, cystinuria, iminoglycinuria, Hartup disease, and Fanconi disease; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; an autoimmune/inflammatory disorder such as such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjögren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus. The polynucleotide sequences encoding SAT may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered SAT expression. Such qualitative or quantitative methods are well known in the art.

[0238] In a particular aspect, the nucleotide sequences encoding SAT may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding SAT may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding SAT in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.

[0239] In order to provide a basis for the diagnosis of a disorder associated with expression of SAT, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding SAT, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

[0240] Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

[0241] With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

[0242] Additional diagnostic uses for oligonucleotides designed from the sequences encoding SAT may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding SAT, or a fragment of a polynucleotide complementary to the polynucleotide encoding SAT, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.

[0243] In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding SAT may be used to detect single nucleotide polymorphisms (SNPs). SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans. Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding SAT are used to amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like. SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels. In fSCCP, the oligonucleotide primers are fluorescently labeled, which allows detection of the amplimers in high-throughput equipment such as DNA sequencing machines. Additionally, sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence. These computer-based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego Calif.).

[0244] Methods which may also be used to quantify the expression of SAT include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.

[0245] In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.

[0246] In another embodiment, SAT, fragments of SAT, or antibodies specific for SAT may be used as elements on a microarray. The microarray may be used to monitor or measure protein-protein interactions, drug-target interactions, and gene expression profiles, as described above.

[0247] A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type. A transcript image represents the global pattern of gene expression by a particular tissue or cell type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time. (See Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No. 5,840,484, expressly incorporated by reference herein.) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type. In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray. The resultant transcript image would provide a profile of gene activity.

[0248] Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples. The transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a cell line.

[0249] Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds. All compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference herein). If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties. These fingerprints or signatures are most useful and refined when they contain expression information from a large number of genes and gene families. Ideally, a genome-wide measurement of expression provides the highest quality signature. Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data. The normalization procedure is useful for comparison of expression data after treatment with different compounds. While the assignment of gene function to elements of a toxicant signature aids in interpretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity. (See, for example, Press Release 00-02 from the National Institute of Environmental Health Sciences, released Feb. 29, 2000, available at http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and desirable in toxicological screening using toxicant signatures to include all expressed gene sequences.

[0250] In one embodiment, the toxicity of a test compound is assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels corresponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.

[0251] Another particular embodiment relates to the use of the polypeptide sequences of the present invention to analyze the proteome of a tissue or cell type. The term proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individually to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a cell's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or cell type. In one embodiment, the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra). The proteins are visualized in the gel as discrete and uniquely positioned spots, typically by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains. The optical density of each protein spot is generally proportional to the level of the protein in the sample. The optical densities of equivalently positioned protein spots from different samples, for example, from biological samples either treated or untreated with a test compound or therapeutic agent, are compared to identify any changes in protein spot density related to the treatment. The proteins in the spots are partially sequenced using, for example, standard methods employing chemical or enzymatic cleavage followed by mass spectrometry. The identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of the present invention. In some cases, further sequence data may be obtained for definitive protein identification.

[0252] A proteomic profile may also be generated using antibodies specific for SAT to quantify the levels of SAT expression. In one embodiment, the antibodies are used as elements on a microarray, and protein expression levels are quantified by exposing the microarray to the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999) Biotechniques 27:778-788). Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each array element.

[0253] Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in parallel with toxicant signatures at the transcript level. There is a poor correlation between transcript and protein abundances for some proteins in some tissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant signatures may be useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile. In addition, the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more reliable and informative in such cases.

[0254] In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.

[0255] In another embodiment, the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated, with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.

[0256] Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.) Various types of microarrays are well known and thoroughly described in DNA Microarrays: A Practical Approach, M. Schena, ed. (1999) Oxford University Press, London, hereby expressly incorporated by reference.

[0257] In another embodiment of the invention, nucleic acid sequences encoding SAT may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization during chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet 7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP). (See, for example, Lander, E. S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357.)

[0258] Fluorescent in situ hybridization (FISH) may be correlated with other physical and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding SAT on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.

[0259] In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the gene or genes responsible for a disease or syndrome have been crudely localized by genetic linkage to a particular genomic region, e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.

[0260] In another embodiment of the invention, SAT, its catalytic or immunogenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between SAT and the agent being tested may be measured.

[0261] Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT application WO84/03564.) In this method, large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with SAT, or fragments thereof, and washed. Bound SAT is then detected by methods well known in the art. Purified SAT can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

[0262] In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding SAT specifically compete with a test compound for binding SAT. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with SAT.

[0263] In additional embodiments, the nucleotide sequences which encode SAT may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

[0264] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

[0265] The disclosures of all patents, applications, and publications mentioned above and below, including U.S. Ser. No. 60/215,465, U.S. Ser. No. 60/239,384, and U.S. Ser. No. 60/253,639, are hereby expressly incorporated by reference.

EXAMPLES

[0266] I. Construction of cDNA Libraries

[0267] Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.) and shown in Table 4, column 5. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCl cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.

[0268] Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase. For most libraries, poly(A)+ RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).

[0269] In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen, Carlsbad Calif.), PBK-CMV plasmid (Stratagene), or pINCY (Incyte Genomics, Palo Alto Calif.), or derivatives thereof. Recombinant plasmids were transformed into competent E. coli cells including XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies.

[0270] II. Isolation of cDNA Clones

[0271] Plasmids obtained as described in Example I were recovered from host cells by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis. Plasmids were purified using at least one of the following: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4° C.

[0272] Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V. B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland).

[0273] III. Sequencing and Analysis

[0274] Incyte cDNA recovered in plasmids as described in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example VIII.

[0275] The polynucleotide sequences derived from Incyte cDNAs were validated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis. The Incyte cDNA sequences or translations thereof were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov model (HMM)-based protein family databases such as PFAM. (HMM is a probabilistic approach which analyzes consensus primary structures of gene families. See, for example, Eddy, S. R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The queries were performed using programs based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences were assembled to produce full length polynucleotide sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences (see Examples IV and V) were used to extend Incyte cDNA assemblages to full length. Assembly was performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length polypeptide sequences. Alternatively, a polypeptide of the invention may begin at any of the methionine residues of the full length translated polypeptide. Full length polypeptide sequences were subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov model (HMM)-based protein family databases such as PFAM. Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco Calif.) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.

[0276] Table 7 summarizes the tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and full length sequences and provides applicable descriptions, references, and threshold parameters. The first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).

[0277] The programs described above for the assembly and analysis of full length polynucleotide and polypeptide sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO:10-18. Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies are described in Table 4, column 4.

[0278] IV. Identification and Editing of Coding Sequences from Genomic DNA

[0279] Putative secretion and trafficking molecules were initially identified by running the Genscan gene identification program against public genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon. The output of Genscan is a FASTA database of polynucleotide and polypeptide sequences. The maximum range of sequence for Genscan to analyze at once was set to 30 kb. To determine which of these Genscan predicted cDNA sequences encode secretion and trafficking molecules, the encoded polypeptides were analyzed by querying against PFAM models for secretion and trafficking molecules. Potential secretion and trafficking molecules were also identified by homology to Incyte cDNA sequences that had been annotated as secretion and trafficking molecules. These selected Genscan-predicted sequences were then compared by BLAST analysis to the genpept and gbpri public databases. Where necessary, the Genscan-predicted sequences were then edited by comparison to the top BLAST hit from genpept to correct errors in the sequence predicted by Genscan, such as extra or omitted exons. BLAST analysis was also used to find any Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus providing evidence for transcription. When Incyte cDNA coverage was available, this information was used to correct or confirm the Genscan predicted sequence. Full length polynucleotide sequences were obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or public cDNA sequences using the assembly process described in Example III. Alternatively, full length polynucleotide sequences were derived entirely from edited or unedited Genscan-predicted coding sequences.

[0280] V. Assembly of Genomic Sequence Data with cDNA Sequence Data

[0281] “Stitched” Sequences

[0282] Partial cDNA sequences were extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III were mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster was analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that were subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval was present on more than one sequence in the cluster were identified, and intervals thus identified were considered to be equivalent by transitivity. For example, if an interval was present on a cDNA and two genomic sequences, then all three intervals were considered to be equivalent. This process allows unrelated but consecutive genomic sequences to be brought together, bridged by cDNA sequence. Intervals thus identified were then “stitched” together by the stitching algorithm in the order that they appear along their parent sequences to generate the longest possible sequence, as well as sequence variants. Linkages between intervals which proceed along one type of parent sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given preference over linkages which change parent type (cDNA to genomic sequence). The resultant stitched sequences were translated and compared by BLAST analysis to the genpept and gbpri public databases. Incorrect exons predicted by Genscan were corrected by comparison to the top BLAST hit from genpept. Sequences were further extended with additional cDNA sequences, or by inspection of genomic DNA, when necessary.

[0283] “Stretched” Sequences

[0284] Partial DNA sequences were extended to full length with an algorithm based on BLAST analysis. First, partial cDNAs assembled as described in Example III were queried against public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program. The nearest GenBank protein homolog was then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV. A chimeric protein was generated by using the resultant high-scoring segment pairs (HSPs) to map the translated sequences onto the GenB ank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog. The GenBank protein homolog, the chimeric protein, or both were used as probes to search for homologous genomic sequences from the public human genome databases. Partial DNA sequences were therefore “stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences were examined to determine whether it contained a complete gene.

[0285] VI. Chromosomal Mapping of SAT Encoding Polynucleotides

[0286] The sequences which were used to assemble SEQ ID NO:10-18 were compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith-Waterman algorithm. Sequences from these databases that matched SEQ ID NO:10-18 were assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Généthon were used to determine if any of the clustered sequences had been previously mapped. Inclusion of a mapped sequence in a cluster resulted in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.

[0287] Map locations are represented by ranges, or intervals, of human chromosomes. The map position of an interval, in centiMorgans, is measured relative to the terminus of the chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.) The cM distances are based on genetic markers mapped by Généthon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI “GeneMap'99” World Wide Web site (http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.

[0288] VII. Analysis of Polynucleotide Expression

[0289] Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and 16.)

[0290] Analogous computer techniques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as: $\frac{{BLAST}\quad {Score} \times {Percent}\quad {Identity}}{5 \times {minimum}\quad \left\{ {{{length}\left( {{Seq}.\quad 1} \right)},{{length}\left( {{Seq}.\quad 2} \right)}} \right\}}$

[0291] The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. The product score is a normalized value between 0 and 100, and is calculated as follows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences). The BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and −4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score. The product score represents a balance between fractional overlap and quality in a BLAST alignment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.

[0292] Alternatively, polynucleotide sequences encoding SAT are analyzed with respect to the tissue sources from which they were derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III). Each cDNA sequence is derived from a cDNA library constructed from a human tissue. Each human tissue is classified into one of the following organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ cells; hemic and immune system; liver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract. The number of libraries in each category is counted and divided by the total number of libraries across all categories. Similarly, each human tissue is classified into one of the following disease/condition categories: cancer, cell line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of libraries in each category is counted and divided by the total number of libraries across all categories. The resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding SAT. cDNA sequences and cDNA library/tissue information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

[0293] VIII. Extension of SAT Encoding Polynucleotides

[0294] Full length polynucleotide sequences were also produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5′ extension of the known fragment, and the other primer was synthesized to initiate 3′ extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68° C. to about 72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations was avoided.

[0295] Selected human cDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.

[0296] High fidelity amplification was obtained by PCR using methods well known in the art. PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺, (NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: 94° C., at 4° C. In the alternative, the parameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min; Step 7: storage at 4° C.

[0297] The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1× TE and 0.5 μl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixture was analyzed by electrophoresis on a 1% agarose gel to determine which reactions were successful in extending the sequence.

[0298] The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison Wis.), and sonicated or sheared prior to religation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega). Extended clones were religated using T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells. Transformed cells were selected on antibiotic-containing media, and individual colonies were picked and cultured overnight at 37° C. in 384-well plates in LB/2× carb liquid media.

[0299] The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).

[0300] In like manner, full length polynucleotide sequences are verified using the above procedure or are used to obtain 5′ regulatory sequences using the above procedure along with oligonucleotides designed for such extension, and an appropriate genomic library.

[0301] IX. Labeling and Use of Individual Hybridization Probes

[0302] Hybridization probes derived from SEQ ID NO:10-18 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).

[0303] The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40° C. To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.

[0304] X. Microarrays

[0305] The linkage or synthesis of array elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra.), mechanical microspotting technologies, and derivatives thereof. The substrate in each of the aforementioned technologies should be uniform and solid with a non-porous surface (Schena (1999), supra). Suggested substrates include silicon, silica, glass slides, glass chips, and silicon wafers. Alternatively, a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures. A typical array may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)

[0306] Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers thereof may comprise the elements of the microarray. Fragments or oligomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). The array elements are hybridized with polynucleotides in a biological sample. The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection. After hybridization, nonhybridized nucleotides from the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each array element. Alternatively, laser desorbtion and mass spectrometry may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microarray may be assessed. In one embodiment, microarray preparation and usage is described in detail below.

[0307] Tissue or Cell Sample Preparation

[0308] Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1× first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μM dGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits (Incyte). Specific control poly(A)⁺ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C. for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto Calif.) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2% SDS.

[0309] Microarray Preparation

[0310] Sequences of the present invention are used to generate array elements. Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts. PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert. Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amplified array elements are then purified using SEPHACRYL400 (Amersham Pharmacia Biotech).

[0311] Purified array elements are immobilized on polymer-coated glass slides. Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester Pa.), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

[0312] Array elements are applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522, incorporated herein by reference. 1 μl of the array element DNA, at an average concentration of 100 ng/μl, is loaded into the open capillary printing element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of array element sample per slide.

[0313] Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microarrays are washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.

[0314] Hybridization

[0315] Hybridization reactions contain 9 μl of sample mixture consisting of 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2% SDS hybridization buffer. The sample mixture is heated to 65° C. for 5 minutes and is aliquoted onto the microarray surface and covered with an 1.8 cm² coverslip. The arrays are transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5×SSC in a corner of the chamber. The chamber containing the arrays is incubated for about 6.5 hours at 60° C. The arrays are washed for 10 min at 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

[0316] Detection

[0317] Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light is focused on the array using a 20× microscope objective (Nikon, Inc., Melville N.Y.). The slide containing the array is placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm array used in the present example is scanned with a resolution of 20 micrometers.

[0318] In two separate scans, a mixed gas multiline laser excites the two fluorophores sequentially. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source. although the apparatus is capable of recording the spectra from both fluorophores simultaneously.

[0319] The sensitivity of the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration. A specific location on the array contains a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. When two samples from different sources (e.g., representing test and control cells), each labeled with a different fluorophore, are hybridized to a single array for the purpose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.

[0320] The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood Mass.) installed in an IBM-compatible PC computer. The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.

[0321] A grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element of the grid. The fluorescence signal within each element is then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte).

[0322] XI. Complementary Polynucleotides

[0323] Sequences complementary to the SAT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring SAT. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of SAT. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the SAT-encoding transcript.

[0324] XII. Expression of SAT

[0325] Expression and purification of SAT is achieved using bacterial or virus-based expression systems. For expression of SAT in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express SAT upon induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of SAT in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autographica californica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding SAT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription. Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)

[0326] In most expression systems, SAT is synthesized as a fusion protein with, e.g., glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST moiety can be proteolytically cleaved from SAT at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch. 10 and 16). Purified SAT obtained by these methods can be used directly in the assays shown in Examples XVI and XVII, where applicable.

[0327] XIII. Functional Assays

[0328] SAT function is assessed by expressing the sequences encoding SAT at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression. Vectors of choice include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad Calif.), both of which contain the cytomegalovirus promoter. 5-10 μg of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation. 1-2 μg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify transfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New York N.Y.

[0329] The influence of SAT on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding SAT and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on the surface of transfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding SAT and other genes of interest can be analyzed by northern analysis or microarray techniques.

[0330] XIV. Production of SAT Specific Antibodies

[0331] SAT substantially purified using polyacrylamide gel electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.

[0332] Alternatively, the SAT amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)

[0333] Typically, oligopeptides of about 15 residues in length are synthesized using an ABI 431A peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-SAT activity by, for example, binding the peptide or SAT to a substrate, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.

[0334] XV. Purification of Naturally Occurring SAT Using Specific Antibodies

[0335] Naturally occurring or recombinant SAT is substantially purified by immunoaffinity chromatography using antibodies specific for SAT. An immunoaffinity column is constructed by covalently coupling anti-SAT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

[0336] Media containing SAT are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of SAT (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/SAT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and SAT is collected.

[0337] XVI. Identification of Molecules which Interact with SAT

[0338] SAT, or biologically active fragments thereof, are labeled with ¹²⁵I Bolton-Hunter reagent. (See, e.g., Bolton A. E. and W. M. Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled SAT, washed, and any wells with labeled SAT complex are assayed. Data obtained using different concentrations of SAT are used to calculate values for the number, affinity, and association of SAT with the candidate molecules.

[0339] Alternatively, molecules interacting with SAT are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or using commercially available kits based on the two-hybrid system, such as the MATCHMAKER system (Clontech).

[0340] SAT may also be used in the PATHCALLING process (CuraGen Corp., New Haven Conn.) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan, K. et al. (2000) U.S. Pat. No. 6,057,101).

[0341] XVII. Demonstration of SAT Activity

[0342] SAT activity is measured by its inclusion in coated vesicles. SAT can be expressed by transforming a mammalian cell line such as COS7, HeLa, or CHO with an eukaryotic expression vector encoding SAT. Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A small amount of a second plasmid, which expresses any one of a number of marker genes, such as β-galactosidase, is co-transformed into the cells in order to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of SAT and β-galactosidase.

[0343] Transformed cells are collected and cell lysates are assayed for vesicle formation. A non-hydrolyzable form of GTP, GTPYS, and an ATP regenerating system are added to the lysate and the mixture is incubated at 37° C. for 10 minutes. Under these conditions, over 90% of the vesicles remain coated (Orci, L. et al. (1989) Cell 56:357-368). Transport vesicles are salt-released from the Golgi membranes, loaded under a sucrose gradient, centrifuged, and fractions are collected and analyzed by SDS-PAGE. Co-localization of SAT with clathrin or COP coatamer is indicative of SAT activity in vesicle formation. The contribution of SAT in vesicle formation can be confirmed by incubating lysates with antibodies specific for SAT prior to GTPγS addition. The antibody will bind to SAT and interfere with its activity, thus preventing vesicle formation.

[0344] In the alternative, SAT activity is measured by its ability to alter vesicle trafficking pathways. Vesicle trafficking in cells transformed with SAT is examined using fluorescence microscopy. Antibodies specific for vesicle coat proteins or typical vesicle trafficking substrates such as transferrin or the mannose-6-phosphate receptor are commercially available. Various cellular components such as ER, Golgi bodies, peroxisomes, endosomes, lysosomes, and the plasmalemma are examined. Alterations in the numbers and locations of vesicles in cells transformed with SAT as compared to control cells are characteristic of SAT activity.

[0345] Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims. TABLE 1 Incyte Incyte Incyte Polypeptide Polypeptide Polynucleotide Polynucleotide Project ID SEQ ID NO: ID SEQ ID NO: ID 1577952 1 1577952CD1 10 1577952CB1 4983705 2 4983705CD1 11 4983705CB1 1310465 3 1310465CD1 12 1310465CB1 4291779 4 4291779CD1 13 4291779CB1 4728247 5 4728247CD1 14 4728247CB1 7472259 6 7472259CD1 15 7472259CB1 7476740 7 7476740CD1 16 7476740CB1 7473774 8 7473774CD1 17 7473774CB1 7946329 9 7946329CD1 18 7946329CB1

[0346] TABLE 2 Incyte GenBank Polypeptide Polypeptide ID Probability SEQ ID NO: ID NO: Score GenBank Homolog 2 4983705CD1 g3077703 2.90E−136 [Oryctolagus cuniculus] mitsugumin29 Takeshima, H. et al. (1998) Biochem. J. 331:317-322. 3 1310465CD1 g777776 0 [Rattus norvegicus] apical endosomal glycoprotein Speelman, B.A. et al. (1995) J. Biol. Chem. 270:1583-1588 4 4291779CD1 g3378150 2.30E−45  [Trypanosoma brucei rhodesiense] lysosomal/endosomal membrane protein p67 Kelley, R.J. et al. (1999) Mol. Biochem. Parasitol. 98:17-28 5 4728247CD1 g5926736 0 [Mus musculus] granuphilin-a Wang, J. et al. (1999) J. Biol. Chem. 274:28542-28548 6 7472259CD1 g8925888 3.70E−101 [Rattus norvegicus] RIM binding protein 1A Wang, Y. et al. (2000) J. Biol. Chem. 275:20033-20044 7 7476740CD1 g8977950 5.60E−43  [Schizosaccharomyces pombe] putative v- snare binding protein; UBA domain 8 7473774CD1 g484296 0 [Rattus norvegicus] Synaptotagmin III Mizuta, M. et al. (1994) J. Biol. Chem. 269:11675-11678 9 7946329CD1 g6136794 1.30E−221 [Mus musculus] synaptotagmin XI Fukuda, M. et al. (1999) J. Biol. Chem. 274:31421-31427

[0347] TABLE 3 SEQ Incyte Amino Potential Potential Analytical ID Polypeptide Acid Phosphorylation Glycosylation Signature Sequences, Methods and NO: ID Residues Sites Sites Domains and Motifs Databases 1 1577952CD1 315 signal_peptide:M1-A33 HMMER Transmembrane domain:S101-L121 HMMER Rhomboid family: HMMER_PFAM Rhomboid:C59-M214 2 4983705CD1 272 S101 S14 S2 N213 SYNAPTOPHYSIN/SYNAPTOPORIN: BLAST_DOMO S262 DM02999|P20488|21-148:G36-V166 SYNAPTOPHYSIN: BLAST_PRODOM PD005837:R27-A268 Synaptophysin/synaptoporin: BLIMPS_BLOCKS BL00604A:L41-L95 BL00604B:T104-L133 BL00604C:V134-L165 BL00604E:C201-Q242 SYNAPTOPHYSIN/SYNAPTOPORIN: BLIMPS_PRINTS PR00220A:I38-T60 PR00220B:A62-H87 PR00220C:F117-N141 PR00220D:F149-G172 PR00220E:V216-F234 Synaptophysin/synaptoporin PROFILESCAN signature: synaptop.prf:Q42-I89 Transmembrane domains: HMMER P114-Y136, I214-F234 Synaptophysin/synaptoporin: HMMER_PFAM Synaptophysin:R27-Q272 3 1310465CD1 1217 S1103 S1183 N204 N282 Signal peptide: M1-A23 SPScan S1188 S140 S318 N340 N584 Signal peptide: M1-G21 HMMER S329 S350 S451 N637 N836 Transmembrane domain: V1156-G1174 HMMER S457 S518 S815 MAM domains: HMMER-PFAM S897 T118 T133 C67-L223, T272-P426, T494-P645, T188 T206 T272 C657-A810, C814-Q970, C974-Q1139 T494 T639 T649 Low-density lipoprotein receptor HMMER-PFAM T739 T863 T877 domains: T903 A228-R268, D456-T493 LDL-receptor class A BL01209: BLIMPS-BLOCKS C249-E261 Low density lipoprotein domains BLIMPS-PRINTS PR00261: K240-E261 MAM domain signatures PR000020: BLIMPS-PRINTS D662-E680, Y113-L129, E724-P735, V1099-A1113, G791-R804 MAN domain: DM01344|A55620|961-1128: BLAST-DOMO L958-L1126, L798-G950, P64-L213, S656-G791, D257-G414 MAM domain: DM01344|A55620|618-796: BLAST-DOMO R616-G794, C814-G950, Q970-G1117, A66-T187, E486-D624 MAN domain: DM01344|A55620|798-959: BLAST-DOMO T795-A957, V945-G1117, G634-A797, C67-A212, D273-L402 MAM domain: DM01344|A55620|300-418: BLAST-DOMO P299-L418, A842-L961, P684-V801, P100-L216 Apical endosomal glycoprotein BLAST-PRODOM pD140389:H269-V655, C230-V427 PD108201:E1130-P1217 PD108200:M1-F65 4 4291779CD1 589 S157 S172 S308 N110 N231 Signal peptide: M1-A41 SPSCAN S456 S506 T208 N436 N465 F09B12.3 protein BLAST-PRODOM T361 T399 Y196 N515 N88 PD145700:Q403-D589 F09B12.3 protein, LANA BLAST-PRODOM lysosomal/endosomal P67 PD043621:C244-D532 Protein splicing site: L258-T263 MOTIFS 5 4728247CD1 671 S11 S139 S147 N537 N579 N97 C2 domains: HMMER-PFAM S190 S193 S213 L373-E462, L528-R617 S217 S231 S235 C2 domain signatures/profiles: PROFILESCAN S274 S289 S363 E498-M570, I360-K416 S393 S414 S467 Synaptogamin signatures PR00399: BLIMPS-PRINTS S494 S502 S520 I360-V375, P433-H448 S55 S563 S59 C2 domain signatures PR00360: BLIMPS-PRINTS S625 S635 S652 A543-L555, K572-N585 S74 T115 T131 C2-domain DM00150|P40748|294-423: BLAST-DOMO T151 T212 T262 G358-E460, E527-L618 T357 T413 T419 C2-domain DM00150|JC2473|249-373: BLAST-DOMO T427 T567 T600 G358-G459, L528-D634 T636 T79 T99 C2-domain DM00150|P46097|140-266: BLAST-DOMO Y120 G358-G482, E524-E639 C2-domain DM00150|P24507|233-362: BLAST-DOMO G358-E460, E527-L618 6 7472259CD1 1519 S104 S1043 T843 N664 N883 SH3 domain: HMMER-PFAM S1104 T685 T740 N890 K715-1777 S1114 T390 T442 Leucine zipper motifs: MOTIFS S1135 S1142 T57 L253-L274, L260-L281, L297-L318 S1182 T273 S897 Src homology 3 (SH3) domain: BLOCKS-BLIMPS S1312 S832 S813 D763-Q776 S1443 S152 S163 Wilm's tumour protein signature BLIMPS-PRINTS T1450 S178 S186 PR00049:R203-P217 T1405 S193 S202 T1273 S206 S225 T1199 S412 S568 T1086 S600 S628 T1080 8656 S660 T1026 S665 S687 7 7476740CD1 396 S106 S319 S374 Retroviral aspartyl protease: HMMER-PFAM S380 S387 S391 Y245-A271, Q315-L344 T295 DDI1 (DNA damage inducible protein): BLAST-PRODOM PD017951:A203-H386 8 7473774CD1 590 S160 S166 S240 N365 N436 PROTEIN KINASE C C2 REGION BLAST_DOMO S245 S385 S393 DM03932|P40748|1-292:M1-G290 S247 S248 S584 C2-DOMAIN DM00150 BLAST_DOMO S75 T184 T223 |P40748|294-423:G297-G426, A430-L554 T487 T493 T497 |P40748|425-552:S427-A555, A298-V423 |P24507|364-491:S427-N556, A298-V423 SYNAPTOTAGMIN TRANSMEMBRANE REPEAT BLAST_PRODOM SYNAPSE III SYTIII VI C SYNAPTIC VESICLE PD022173:F142-Q315 SYNAPTOTAGMIN TRANSMEMBRANE REPEAT BLAST_PRODOM SYNAPSE III SYTIII C SYNAPTIC VESICLE PROTEIN PD012608:M1-P102 PD007398:R536-S580 PROTEIN C REPEAT SYNAPTOTAGMIN BLAST_PRODOM PHOSPHOLIPASE TRANSMEMBRANE SYNAPSE BINDING PHORBOLESTER KINASE PD000136:L316-Q400, L448-C535 C2 domain proteins BLIMPS_PFAM PF00168:L458-D468, L475-E500 SYNAPTOTAGMIN SIGNATURE BLIMPS_PRINTS PR00399:P373-D388, 5393-L403, I303-V318, V318-S331 C2 domain signature BLIMPS_PRINTS PR00360:S331-L343, K358-S371, L382-D390 Transmembrane domain:I51-V74 HMMER C2 domain C2:L316-V402, L448-R536 HMMER_PFAM C2 domain signature gcg_motif: MOTIFS A323-Y338 A455-Y470 C2 domain signature and profile PROFILESCAN c2_domain.prf:L435-K490, I303-K358 9 7946329CD1 431 S117 S150 S151 N360 C2-DOMAIN DM00150 BLAST_DOMO S253 S291 S406 |I59355|151-280:L158-N285, G293-S406 S417 S427 S70 |P40749|151-280:L158-N285, G293-S406 T140 T177 T272 |I59355|282-412:Q287-R419 T348 T354 T387 |P40749|282-412:Q287-R419 T388 SYNAPTOTAGMIN IV TRANSMEMBRANE BLAST_PRODOM REPEAT SYNAPSE XI PD022115:M1-V175 PD151917:E260-Q302 SYNAPTOTAGMIN XI BLAST_PRODOM PD163942:L396-Y431 PROTEIN C REPEAT SYNAPTOTAGMIN BLAST_PRODOM PHOSPHOLIPASE TRANSMEMBRANE SYNAPSE BINDING PHORBOLESTER KINASE PD000136:L174-E260, M308-E391 SYNAPTOTAGMIN SIGNATURE BLIMPS_PRINTS PR00399:V310-I323, P233-S248, S253-V263, L161-V176 C2 domain signature BLIMPS_PRINTS PR00360:Q190-I202, K217-Y230, L242-D250 Transmembrane domain: V16-W37 HMMER C2 domain C2: HMMER_PFAM L174-M262, M308-I397 C2 domain signature and profile PROFILESCAN c2_domain.prf: L295-K351, L161-K217 Spscan signal_cleavage:M1-S38 SPSCAN

[0348] TABLE 4 Incyte Polynucleotide Polynucleotide Sequence Selected 5′ 3′ SEQ ID NO: ID Length Fragments Sequence Fragments Position Position 10 1577952CB1 3424  1-39 70687032V1 2938 3424 2099-2292 7438458H1 (ADRETUE02) 2439 3019 3272-3424 71111635V1 574 1240 70647387V1 2364 2984 8009276H1 (NOSEDIC02) 1529 2189 71112240V1 665 1360 71114546V1 1294 1881 7993001H1 (UTRSDIC01) 1906 2435 70767606V1 1 627 1438701F1 (PANCNOT08) 1361 1899 11 4983705CB1 1033  732-1033 GBI:g6524214_000026.raw.1 674 1033 g5810426 1 445 49837Q5H1 (HELATXT05) 266 535 6901767R8 (MUSLTDR02) 291 867 7610385H1 (KIDCTME01) 39 305 12 1310465CB1 3902   1-1452 7164475F8 (PLACNOR01) 416 1016 1764-2846 71991186V1 2676 3281 7714340H1 (SINTFEE02) 1136 1732 7714340J1 (SINTFEE02) 1433 2056 8117813H1 (TONSDIC01) 2011 2714 6542726H1 (LNODNON02) 3386 3902 71987767V1 2777 3431 71991460V1 2136 2767 7080102F6 (STOMTMR02) 466 1222 7693543H2 (LNODTUE01) 1 438 13 4291779CB1 2574  979-1592 70735272V1 1460 2082 2526437F6 (BRAITUT21) 1720 2198 6154207H1 (ENDMUNT04) 1341 1668 6996766H1 (BRAXTDR17) 635 1150 7061302H1 (PENITMN02) 946 1663 1869872F6 (SKINBIT01) 2185 2574 70738661V1 1973 2557 7278956H1 (BMARTXE01) 1 565 4822574F9 (PROSTUT17) 479 937 14 4728247CB1 2878  539-2368 2079722F6 (UTRSNOT08) 1964 2462 70867423V1 2297 2878 71171848V1 1383 1966 71399565V1 641 1331 224668R6 (PANCNOT01) 369 868 3354730F6 (PROSNOT28) 1 579 224668T6 (PANCNOT01) 1831 2455 7631669H1 (BRAFTUE03) 1309 1902 15 7472259CB1 5628   1-2080 7396961H1 (KIDEUNE02) 1374 2096 5302-5628 7761273H1 (THYMNOE02) 1 498 2575-4411 6842194H1 (BRSTNON02) 4134 4726 2394503F6 (THP1AZT01) 5028 5628 g1963660 4411 4860 g2063588 4767 5235 GNN.g6006353_010 1 4922 16 7476740CB1 1482  1-566 GNN.g7547222₋000015_002 175 1365 750-831 g2064117 946 1482 6247775F8 (TESTNOT17) 1 755 17 7473774CB1 2511  1-113 70555497V1 1470 2036  639-1330 72049138V1 716 1538 2471-2511 7585860H2 (BRAIFEC01) 1 630 71045227V1 1640 2282 72050239V1 587 1476 71539112V1 2184 2511 18 7946329CB1 1680 7228832H1 (BRAXTDR15) 1 480 71801792V1 460 1167 71801805V1 607 1231 6996340H1 (BRAXTDR17) 1091 1680

[0349] TABLE 5 Polynucleotide Incyte Project Representative SEQ ID NO: ID Library 10 1577952CB1 LIVRTUT13 11 4983705CB1 BRAZNOT01 12 1310465CB1 SINTFEE02 13 4291779CB1 BRAITUT21 14 4728247CB1 PANCNOT01 15 7472259CB1 THP1AZT01 16 7476740CB1 TESTNOT17 17 7473774CB1 BRAIFEC01 18 7946329CB1 SCOMDIC01

[0350] TABLE 6 Library Vector Library Description BRAIFEC01 pINCY This large size-fractionated library was constructed using RNA isolated from brain tissue removed from a Caucasian male fetus who was stillborn with a hypoplastic left heart at 23 weeks' gestation. BRAITUT21 pINCY Library was constructed using RNA isolated from brain tumor tissue removed from the midline frontal lobe of a 61-year-old Caucasian female during excision of a cerebral meningeal lesion. Pathology indicated subfrontal meningothelial meningioma with no atypia. One ethmoid and mucosal tissue sample indicated meningioma. Family history included cerebrovascular disease, senile dementia, hyperlipidemia, benign hypertension, atherosclerotic coronary artery disease, congestive heart failure, and breast cancer. BRAZNOT01 pINCY Library was constructed using RNA isolated from striatum, globus pallidus and posterior putamen tissue removed from a 45-year-old Caucasian female who died from a dissecting aortic aneurysm and ischemic bowel disease. Pathology indicated mild arteriosclerosis involving the cerebral cortical white matter and basal ganglia. Grossly, there was mild meningeal fibrosis and mild focal atherosclerotic plaque in the middle cerebral artery, as well as vertebral arteries bilaterally. Microscopically, the cerebral hemispheres, brain stem and cerebellum revealed focal areas in the white matter that contained blood vessels that were barrel-shaped, hyalinized, with hemosiderin-laden macrophages in the Virchow-Robin space. In addition, there were scattered neurofibrillary tangles within the basolateral nuclei of the amygdala. Patient history included mild atheromatosis of aorta and coronary arteries, bowel and liver infarct due to aneurysm, physiologic fatty liver associated with obesity, mild diffuse emphysema, thrombosis of mesenteric and portal veins, cardiomegaly due to hypertrophy of left ventricle, arterial hypertension, acute pulmonary edema, splenomegaly, obesity (300 lb.), leiomyoma of uterus, sleep apnea, and iron deficiency anemia. LIVRTUT13 pINCY Library was constructed using RNA isolated from liver tumor tissue removed from a 62-year-old Caucasian female during partial hepatectomy and exploratory laparotomy. Pathology indicated metastatic intermediate grade neuroendocrine carcinoma, consistent with islet cell tumor, forming nodules ranging in size, in the lateral and medial left liver lobe. The pancreas showed fibrosis, chronic inflammation and fat necrosis consistent with pseudocyst. The gall bladder showed mild chronic cholecystitis. Patient history included malignant neoplasm of the pancreas tail, pulmonary embolism, hyperlipidemia, thrombophlebitis, joint pain in multiple joints, type II diabetes, benign hypertension, and cerebrovascular disease. Family history included pancreas cancer, secondary liver cancer, benign hypertension, and hyperlipidemia. PANCNOT01 PBLUESCRIPT Library was constructed using RNA isolated from the pancreatic tissue of a 29- year-old Caucasian male who died from head trauma. SCOMDIC01 PSPORT1 This large size-fractionated library was constructed using RNA isolated from diseased spinal cord tissue removed from the base of the medulla of a 57-year- old Caucasian male, who died from a cerebrovascular accident. Serologies were negative. Patient history included Huntington's disease, emphysema, and tobacco abuse. (3-4 packs per day, for 40 years). SINTFEE02 PCDNA2.1 This 5′ biased random primed library was constructed using RNA isolated from small intestine tissue removed from a Caucasian male fetus who died from Patau's syndrome (trisomy 13) at 20-weeks' gestation. Serology was negative. TESTNOT17 pINCY Library was constructed from testis tissue removed from a 26-year-old Caucasian male who died from head trauma due to a motor vehicle accident. Serologies were negative. Patient history included a hernia at birth, tobacco use (1½ ppd), marijuana use, and daily alcohol use (beer and hard liquor). THP1AZT01 pINCY Library was constructed using RNA isolated from THP-1 promonocyte cells treated for three days with 0.8 micromolar 5-aza-2′-deoxycytidine. THP-1 (ATCC TIB 202) is a human promonocyte line derived from peripheral blood of a 1-year-old Caucasian male with acute monocytic leukemia (Int. J. Cancer (1980) 26:171)

[0351] TABLE 7 Parameter Program Description Reference Threshold ABI FACTURA A program that removes vector sequences and Applied Biosystems, Foster City, CA. masks ambiguous bases in nucleic acid sequences. ABI/PARACEL A Fast Data Finder useful in comparing and Applied Biosystems, Foster City, CA; Mismatch <50% FDF annotating amino acid or nucleic acid sequences. Paracel Inc., Pasadena, CA. ABI A program that assembles nucleic acid sequences. Applied Biosystems, Foster City, CA. AutoAssembler BLAST A Basic Local Alignment Search Tool useful in Altschul, S. F. et al. (1990) J. Mol. Biol. ESTs: Probability sequence similarity search for amino acid and 215:403-410; Altschul, S. F. et al. (1997) value = 1.0E−8 nucleic acid sequences. BLAST includes five Nucleic Acids Res. 25:3389-3402. or less functions: blastp, blastn, blastx, tblastn, and tblastx. Full Length sequences: Probability value = 1.0E−10 or less FASTA A Pearson and Lipman algorithm that searches for Pearson, W. R. and D. J. Lipman (1988) Proc. ESTs: fasta E similarity between a query sequence and a group of Natl. Acad Sci. USA 85:2444-2448; Pearson, value = 1.06E−6 sequences of the same type. FASTA comprises as W.R. (1990) Methods Enzymol. 183:63-98; Assembled ESTs: least five functions: fasta, tfasta, fastx, tfastx, and and Smith, T. F. and M. S. Waterman (1981) fasta Identity = ssearch. Adv. Appl. Math. 2:482-489. 95% or greater and Match length = 200 bases or greater; fastx E value = 1.0E−8 or less Full Length sequences: fastx score = 100 or greater BLIMPS A BLocks IMProved Searcher that matches a Henikoff, S. and J. G. Henikoff (1991) Nucleic Probability sequence against those in BLOCKS, PRINTS, Acids Res. 19:6565-6572; Henikoff, J. G. and value = 1.0E−3 DOMO, PRODOM, and PFAM databases to search S. Henikoff (1996) Methods Enzymol. or less for gene families, sequence homology, and 266:88-105; and Attwood, T.K. et al. (1997) J. structural fingerprint regions. Chem. Inf. Comput. Sci. 37:417-424. HMMER An algorithm for searching a query sequence against Krogh, A. et al. (1994) J. Mol. Biol. PFAM hits: hidden Markov model (HMM)-based databases of 235:1501-1531; Sonnhammer, E. L. L, et al. Probability protein family consensus sequences, such as PFAM. (1988) Nucleic Acids Res. 26:320-322; value = 1.0E−3 Durbin, R. et al. (1998) Our World View, in a or less Nutshell, Cambridge Univ. Press, pp. 1-350. Signal peptide hits. Score = 0 or greater ProfileScan An algorithm that searches for structural and sequence Gribskov, M. et al. (1988) CABIOS 4:61-66; Normalized motifs in protein sequences that match sequence patterns Gribskov, M. et al. (1989) Methods Enzymol. quality score ≧ defined in Prosite. 183:146-159; Bairoch, A. et al. (1997) GCG-specified Nucleic Acids Res. 25:217-221. “HIGH” value for that particular Prosite motif. Generally, score = 1.4-2.1. Phred A base-calling algorithm that examines automated Ewing, B. et al. (1998) Genome Res. sequencer traces with high sensitivity and probability. 8:175-185; Ewing, B. and P. Green (1998) GenomeRes. 8:186-194. Phrap A Phils Revised Assembly Program including SWAT and Smith, T. F. and M. S. Waterman (1981) Adv. Score = 120 or CrossMatch, programs based on efficient implementation Appl. Math. 2:482-489; Smith, T. F. and M. S. greater; Match of the Smith-Waterman algorithm, useful in searching Waterman (1981) J. Mol. Biol. 147:195-197; length = 56 sequence homology and assembling DNA sequences. and Green, P., University of Washington, or greater Seattle, WA. Consed A graphical tool for viewing and editing Phrap Gordon, D. et al. (1998) Genome Res. 8:195-202. assemblies. SPScan A weight matrix analysis program that scans protein Nielson, H. et al. (1997) Protein Engineering Score = 3.5 sequences for the presence of secretory signal peptides. 10:1-6; Claverie, J. M. and S. Audic (1997) or greater CABIOS 12:431-439. TMAP A program that uses weight matrices to delineate Persson, B. and P. Argos (1994) J. Mol. Biol, transmembrane segments on protein sequences and 237:182-192; Persson, B. and P. Argos (1996) determine orientation. Protein Sci. 5:363-371. TMHMMER A program that uses a hidden Markov model (HMM) to Sonnhammer, E. L. et al. (1998) Proc. Sixth Intl. delineate transmembrane segments on protein sequences Conf. on Intelligent Systems for Mol. Biol., and determine orientation. Glasgow et al., eds., The Am. Assoc. for Artificial Intelligence Press, Menlo Park, CA, pp. 175-182. Motifs A program that searches amino acid sequences for Bairoch, A. et al. (1997) Nucleic Acids Res. patterns that matched those defined in Prosite. 25:217-221; Wisconsin Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI.

[0352]

1 18 1 315 PRT Homo sapiens misc_feature Incyte ID No 1577952CD1 1 Met Gln Arg Arg Ser Arg Gly Ile Asn Thr Gly Leu Ile Leu Leu 1 5 10 15 Leu Ser Gln Ile Phe His Val Gly Ile Asn Asn Ile Pro Pro Val 20 25 30 Thr Leu Ala Thr Leu Ala Leu Asn Ile Trp Phe Phe Leu Asn Pro 35 40 45 Gln Lys Pro Leu Tyr Ser Ser Cys Leu Ser Val Glu Lys Cys Tyr 50 55 60 Gln Gln Lys Asp Trp Gln Arg Leu Leu Leu Ser Pro Leu His His 65 70 75 Ala Asp Asp Trp His Leu Tyr Phe Asn Met Ala Ser Met Leu Trp 80 85 90 Lys Gly Ile Asn Leu Glu Arg Arg Leu Gly Ser Arg Trp Phe Ala 95 100 105 Tyr Val Ile Thr Ala Phe Ser Val Leu Thr Gly Val Val Tyr Leu 110 115 120 Leu Leu Gln Phe Ala Val Ala Glu Phe Met Asp Glu Pro Asp Phe 125 130 135 Lys Arg Ser Cys Ala Val Gly Phe Ser Gly Val Leu Phe Ala Leu 140 145 150 Lys Val Leu Asn Asn His Tyr Cys Pro Gly Gly Phe Val Asn Ile 155 160 165 Leu Gly Phe Pro Val Pro Asn Arg Phe Ala Cys Trp Val Glu Leu 170 175 180 Val Ala Ile His Leu Phe Ser Pro Gly Thr Ser Phe Ala Gly His 185 190 195 Leu Ala Gly Ile Leu Val Gly Leu Met Tyr Thr Gln Gly Pro Leu 200 205 210 Lys Lys Ile Met Glu Ala Cys Ala Gly Gly Phe Ser Ser Ser Val 215 220 225 Gly Tyr Pro Gly Arg Gln Tyr Tyr Phe Asn Ser Ser Gly Ser Ser 230 235 240 Gly Tyr Gln Asp Tyr Tyr Pro His Gly Arg Pro Asp His Tyr Glu 245 250 255 Glu Ala Pro Arg Asn Tyr Asp Thr Tyr Thr Ala Gly Leu Ser Glu 260 265 270 Glu Glu Gln Leu Glu Arg Ala Leu Gln Ala Ser Leu Trp Asp Arg 275 280 285 Gly Asn Thr Arg Asn Ser Pro Pro Pro Tyr Gly Phe His Leu Ser 290 295 300 Pro Glu Glu Met Arg Arg Gln Arg Leu His Arg Phe Asp Ser Gln 305 310 315 2 272 PRT Homo sapiens misc_feature Incyte ID No 4983705CD1 2 Met Ser Ser Thr Glu Ser Ala Gly Arg Thr Ala Asp Lys Ser Pro 1 5 10 15 Arg Gln Gln Val Asp Arg Leu Leu Val Gly Leu Arg Trp Arg Arg 20 25 30 Leu Glu Glu Pro Leu Gly Phe Ile Lys Val Leu Gln Trp Leu Phe 35 40 45 Ala Ile Phe Ala Phe Gly Ser Cys Gly Ser Tyr Ser Gly Glu Thr 50 55 60 Gly Ala Met Val Arg Cys Asn Asn Glu Ala Lys Asp Val Ser Ser 65 70 75 Ile Ile Val Ala Phe Gly Tyr Pro Phe Arg Leu His Arg Ile Gln 80 85 90 Tyr Glu Met Pro Leu Cys Asp Glu Glu Ser Ser Ser Lys Thr Met 95 100 105 His Leu Met Gly Asp Phe Ser Ala Pro Ala Glu Phe Phe Val Thr 110 115 120 Leu Gly Ile Phe Ser Phe Phe Tyr Thr Met Ala Ala Leu Val Ile 125 130 135 Tyr Leu Arg Phe His Asn Leu Tyr Thr Glu Asn Lys Arg Phe Pro 140 145 150 Leu Val Asp Phe Cys Val Thr Val Ser Phe Thr Phe Phe Trp Leu 155 160 165 Val Ala Ala Ala Ala Trp Gly Lys Gly Leu Thr Asp Val Lys Gly 170 175 180 Ala Thr Arg Pro Ser Ser Leu Thr Ala Ala Met Ser Val Cys His 185 190 195 Gly Glu Glu Ala Val Cys Ser Ala Gly Ala Thr Pro Ser Met Gly 200 205 210 Leu Ala Asn Ile Ser Val Leu Phe Gly Phe Ile Asn Phe Phe Leu 215 220 225 Trp Ala Gly Asn Cys Trp Phe Val Phe Lys Glu Thr Pro Trp His 230 235 240 Gly Gln Gly Gln Gly Gln Asp Gln Asp Gln Asp Gln Asp Gln Gly 245 250 255 Gln Gly Pro Ser Gln Glu Ser Ala Ala Glu Gln Gly Ala Val Glu 260 265 270 Lys Gln 3 1217 PRT Homo sapiens misc_feature Incyte ID No 1310465CD1 3 Met Pro Leu Ser Ser His Leu Leu Pro Ala Leu Val Leu Phe Leu 1 5 10 15 Ala Ala Gly Ser Ser Gly Trp Ala Trp Val Pro Asn His Cys Arg 20 25 30 Ser Pro Gly Gln Ala Val Cys Asn Phe Val Cys Asp Cys Arg Asp 35 40 45 Cys Ser Asp Glu Ala Gln Cys Gly Tyr His Gly Ala Ser Pro Thr 50 55 60 Leu Gly Ala Pro Phe Ala Cys Asp Phe Glu Gln Asp Pro Cys Gly 65 70 75 Trp Arg Asp Ile Ser Thr Ser Gly Tyr Ser Trp Leu Arg Asp Arg 80 85 90 Ala Gly Ala Ala Leu Glu Gly Pro Gly Pro His Ser Asp His Thr 95 100 105 Leu Gly Thr Asp Leu Gly Trp Tyr Met Ala Val Gly Thr His Arg 110 115 120 Gly Lys Glu Ala Ser Thr Ala Ala Leu Arg Ser Pro Thr Leu Arg 125 130 135 Glu Ala Ala Ser Ser Cys Lys Leu Arg Leu Trp Tyr His Ala Ala 140 145 150 Ser Gly Asp Val Ala Glu Leu Arg Val Glu Leu Thr His Gly Ala 155 160 165 Glu Thr Leu Thr Leu Trp Gln Ser Thr Gly Pro Trp Gly Pro Gly 170 175 180 Trp Gln Glu Leu Ala Val Thr Thr Gly Arg Ile Arg Gly Asp Phe 185 190 195 Arg Val Thr Phe Ser Ala Thr Arg Asn Ala Thr His Arg Gly Ala 200 205 210 Val Ala Leu Asp Asp Leu Glu Phe Trp Asp Cys Gly Leu Pro Thr 215 220 225 Pro Gln Ala Asn Cys Pro Pro Gly His His His Cys Gln Asn Lys 230 235 240 Val Cys Val Glu Pro Gln Gln Leu Cys Asp Gly Glu Asp Asn Cys 245 250 255 Gly Asp Leu Ser Asp Glu Asn Pro Leu Thr Cys Gly Arg His Ile 260 265 270 Ala Thr Asp Phe Glu Thr Gly Leu Gly Pro Trp Asn Arg Ser Glu 275 280 285 Gly Trp Ser Arg Asn His Arg Ala Gly Gly Pro Glu Arg Pro Ser 290 295 300 Trp Pro Arg Arg Asp His Ser Arg Asn Ser Ala Gln Gly Ser Phe 305 310 315 Leu Val Ser Val Ala Glu Pro Gly Thr Pro Ala Ile Leu Ser Ser 320 325 330 Pro Glu Phe Gln Ala Ser Gly Thr Ser Asn Cys Ser Leu Val Phe 335 340 345 Tyr Gln Tyr Leu Ser Gly Ser Glu Ala Gly Cys Leu Gln Leu Phe 350 355 360 Leu Gln Thr Leu Gly Pro Gly Ala Pro Arg Ala Pro Val Leu Leu 365 370 375 Arg Arg Arg Arg Gly Glu Leu Gly Thr Ala Trp Val Arg Asp Arg 380 385 390 Val Asp Ile Gln Ser Ala Tyr Pro Phe Gln Ile Leu Leu Ala Gly 395 400 405 Gln Thr Gly Pro Gly Gly Val Val Gly Leu Asp Asp Leu Ile Leu 410 415 420 Ser Asp His Cys Arg Pro Val Ser Glu Val Ser Thr Leu Gln Pro 425 430 435 Leu Pro Pro Gly Pro Arg Ala Pro Ala Pro Gln Pro Leu Pro Pro 440 445 450 Ser Ser Arg Leu Gln Asp Ser Cys Lys Gln Gly His Leu Ala Cys 455 460 465 Gly Asp Leu Cys Val Pro Pro Glu Gln Leu Cys Asp Phe Glu Glu 470 475 480 Gln Cys Ala Gly Gly Glu Asp Glu Gln Ala Cys Gly Thr Thr Asp 485 490 495 Phe Glu Ser Pro Glu Ala Gly Gly Trp Glu Asp Ala Ser Val Gly 500 505 510 Arg Leu Gln Trp Arg Arg Val Ser Ala Gln Glu Ser Gln Gly Ser 515 520 525 Ser Ala Ala Ala Ala Gly His Phe Leu Ser Leu Gln Arg Ala Trp 530 535 540 Gly Gln Leu Gly Ala Glu Ala Arg Val Leu Thr Pro Leu Leu Gly 545 550 555 Pro Ser Gly Pro Ser Cys Glu Leu His Leu Ala Tyr Tyr Leu Gln 560 565 570 Ser Gln Pro Arg Gly Phe Leu Ala Leu Val Val Val Asp Asn Gly 575 580 585 Ser Arg Glu Leu Ala Trp Gln Ala Leu Ser Ser Ser Ala Gly Ile 590 595 600 Trp Lys Val Asp Lys Val Leu Leu Gly Ala Arg Arg Arg Pro Phe 605 610 615 Arg Leu Glu Phe Val Gly Leu Val Asp Leu Asp Gly Pro Asp Gln 620 625 630 Gln Gly Ala Gly Val Asp Asn Val Thr Leu Arg Asp Cys Ser Pro 635 640 645 Thr Val Thr Thr Glu Arg Asp Arg Glu Val Ser Cys Asn Phe Glu 650 655 660 Arg Asp Thr Cys Ser Trp Tyr Pro Gly His Leu Ser Asp Thr His 665 670 675 Trp Arg Trp Val Glu Ser Arg Gly Pro Asp His Asp His Thr Thr 680 685 690 Gly Gln Gly His Phe Val Leu Leu Asp Pro Thr Asp Pro Leu Ala 695 700 705 Trp Gly His Ser Ala His Leu Leu Ser Arg Pro Gln Val Pro Ala 710 715 720 Ala Pro Thr Glu Cys Leu Ser Phe Trp Tyr His Leu His Gly Pro 725 730 735 Gln Ile Gly Thr Leu Arg Leu Ala Met Arg Arg Glu Gly Glu Glu 740 745 750 Thr His Leu Trp Ser Arg Ser Gly Thr Gln Gly Asn Arg Trp His 755 760 765 Glu Ala Trp Ala Thr Leu Ser His Gln Pro Gly Ser His Ala Gln 770 775 780 Tyr Gln Leu Leu Phe Glu Gly Leu Arg Asp Gly Tyr His Gly Thr 785 790 795 Met Ala Leu Asp Asp Val Ala Val Arg Pro Gly Pro Cys Trp Ala 800 805 810 Pro Asn Tyr Cys Ser Phe Glu Asp Ser Asp Cys Gly Phe Ser Pro 815 820 825 Gly Gly Gln Gly Leu Trp Arg Arg Gln Ala Asn Ala Ser Gly His 830 835 840 Ala Ala Trp Gly Pro Pro Thr Asp His Thr Thr Glu Thr Ala Gln 845 850 855 Gly His Tyr Met Val Val Asp Thr Ser Pro Asp Ala Leu Pro Arg 860 865 870 Gly Gln Thr Ala Ser Leu Thr Ser Lys Glu His Arg Pro Leu Ala 875 880 885 Gln Pro Ala Cys Leu Thr Phe Trp Tyr His Gly Ser Leu Arg Ser 890 895 900 Pro Gly Thr Leu Arg Val Tyr Leu Glu Glu Arg Gly Arg His Gln 905 910 915 Val Leu Ser Leu Ser Ala His Gly Gly Leu Ala Trp Arg Leu Gly 920 925 930 Ser Met Asp Val Gln Ala Glu Arg Ala Trp Arg Val Val Phe Glu 935 940 945 Ala Val Ala Ala Gly Val Ala His Ser Tyr Val Ala Leu Asp Asp 950 955 960 Leu Leu Leu Gln Asp Gly Pro Cys Pro Gln Pro Gly Ser Cys Asp 965 970 975 Phe Glu Ser Gly Leu Cys Gly Trp Ser His Leu Ala Gly Pro Gly 980 985 990 Leu Gly Gly Tyr Ser Trp Asp Trp Gly Gly Gly Ala Thr Pro Ser 995 1000 1005 Arg Tyr Pro Gln Pro Pro Val Asp His Thr Leu Gly Thr Glu Ala 1010 1015 1020 Gly His Phe Ala Phe Phe Glu Thr Gly Val Leu Gly Pro Gly Gly 1025 1030 1035 Arg Ala Ala Trp Leu Arg Ser Glu Pro Leu Pro Ala Thr Pro Ala 1040 1045 1050 Ser Cys Leu Arg Phe Trp Tyr His Met Gly Phe Pro Glu His Phe 1055 1060 1065 Tyr Lys Gly Glu Leu Lys Val Leu Leu His Ser Ala Gln Gly Gln 1070 1075 1080 Leu Ala Val Trp Gly Ala Gly Gly His Arg Arg His Gln Trp Leu 1085 1090 1095 Glu Ala Gln Val Glu Val Ala Ser Ala Lys Glu Phe Gln Ile Val 1100 1105 1110 Phe Glu Ala Thr Leu Gly Gly Gln Pro Ala Leu Gly Pro Ile Ala 1115 1120 1125 Leu Asp Asp Val Glu Tyr Leu Ala Gly Gln His Cys Gln Gln Pro 1130 1135 1140 Ala Pro Ser Pro Gly Asn Thr Ala Ala Pro Gly Ser Val Pro Ala 1145 1150 1155 Val Val Gly Ser Ala Leu Leu Leu Leu Met Leu Leu Val Leu Leu 1160 1165 1170 Gly Leu Gly Gly Arg Arg Trp Leu Gln Lys Lys Gly Ser Cys Pro 1175 1180 1185 Phe Gln Ser Asn Thr Glu Ala Thr Ala Pro Gly Phe Asp Asn Ile 1190 1195 1200 Leu Phe Asn Ala Asp Gly Val Thr Leu Pro Ala Ser Val Thr Ser 1205 1210 1215 Asp Pro 4 589 PRT Homo sapiens misc_feature Incyte ID No 4291779CD1 4 Met Val Gly Gln Met Tyr Cys Tyr Pro Gly Ser His Leu Ala Arg 1 5 10 15 Ala Leu Thr Arg Ala Leu Ala Leu Ala Leu Val Leu Ala Leu Leu 20 25 30 Val Gly Pro Phe Leu Ser Gly Leu Ala Gly Ala Ile Pro Ala Pro 35 40 45 Gly Gly Arg Trp Ala Arg Asp Gly Pro Val Pro Pro Ala Ser Arg 50 55 60 Ser Arg Ser Val Leu Leu Asp Val Ser Ala Gly Gln Leu Leu Met 65 70 75 Val Asp Gly Arg His Pro Asp Ala Val Ala Trp Ala Asn Leu Thr 80 85 90 Asn Ala Ile Arg Glu Thr Gly Trp Ala Phe Leu Glu Leu Gly Thr 95 100 105 Ser Gly Gln Tyr Asn Asp Ser Leu Gln Ala Tyr Ala Ala Gly Val 110 115 120 Val Glu Ala Ala Val Ser Glu Glu Leu Ile Tyr Met His Trp Met 125 130 135 Asn Thr Val Val Asn Tyr Cys Gly Pro Phe Glu Tyr Glu Val Gly 140 145 150 Tyr Cys Glu Arg Leu Lys Ser Phe Leu Glu Ala Asn Leu Glu Trp 155 160 165 Met Gln Glu Glu Met Glu Ser Asn Pro Asp Ser Pro Tyr Trp His 170 175 180 Gln Val Arg Leu Thr Leu Leu Gln Leu Lys Gly Leu Glu Asp Ser 185 190 195 Tyr Glu Gly Arg Val Ser Phe Pro Ala Gly Lys Phe Thr Ile Lys 200 205 210 Pro Leu Gly Phe Leu Leu Leu Gln Leu Ser Gly Asp Leu Glu Asp 215 220 225 Leu Glu Leu Ala Leu Asn Lys Thr Lys Ile Lys Pro Ser Leu Gly 230 235 240 Ser Gly Ser Cys Ser Ala Leu Ile Lys Leu Leu Pro Gly Gln Ser 245 250 255 Asp Leu Leu Val Ala His Asn Thr Trp Asn Asn Tyr Gln His Met 260 265 270 Leu Arg Val Ile Lys Lys Tyr Trp Leu Gln Phe Arg Glu Gly Pro 275 280 285 Trp Gly Asp Tyr Pro Leu Val Pro Gly Asn Lys Leu Val Phe Ser 290 295 300 Ser Tyr Pro Gly Thr Ile Phe Ser Cys Asp Asp Phe Tyr Ile Leu 305 310 315 Gly Ser Gly Leu Val Thr Leu Glu Thr Thr Ile Gly Asn Lys Asn 320 325 330 Pro Ala Leu Trp Lys Tyr Val Arg Pro Arg Gly Cys Val Leu Glu 335 340 345 Trp Val Arg Asn Ile Val Ala Asn Arg Leu Ala Ser Asp Gly Ala 350 355 360 Thr Trp Ala Asp Ile Phe Lys Arg Phe Asn Ser Gly Thr Tyr Asn 365 370 375 Asn Gln Trp Met Ile Val Asp Tyr Lys Ala Phe Ile Pro Gly Gly 380 385 390 Pro Ser Pro Gly Ser Arg Val Leu Thr Ile Leu Glu Gln Ile Pro 395 400 405 Gly Met Val Val Val Ala Asp Lys Thr Ser Glu Leu Tyr Gln Lys 410 415 420 Thr Tyr Trp Ala Ser Tyr Asn Ile Pro Ser Phe Glu Thr Val Phe 425 430 435 Asn Ala Ser Gly Leu Gln Ala Leu Val Ala Gln Tyr Gly Asp Trp 440 445 450 Phe Ser Tyr Asp Gly Ser Pro Arg Ala Gln Ile Phe Arg Arg Asn 455 460 465 Gln Ser Leu Val Gln Asp Met Asp Ser Met Val Arg Leu Met Arg 470 475 480 Tyr Asn Asp Phe Leu His Asp Pro Leu Ser Leu Cys Lys Ala Cys 485 490 495 Asn Pro Gln Pro Asn Gly Glu Asn Ala Ile Ser Ala Arg Ser Asp 500 505 510 Leu Asn Pro Ala Asn Gly Ser Tyr Pro Phe Gln Ala Leu Arg Gln 515 520 525 Arg Ser His Gly Gly Ile Asp Val Lys Val Thr Ser Met Ser Leu 530 535 540 Ala Arg Ile Leu Ser Leu Leu Ala Ala Ser Gly Pro Thr Trp Asp 545 550 555 Gln Val Pro Pro Phe Gln Trp Ser Thr Ser Pro Phe Ser Gly Leu 560 565 570 Leu His Met Gly Gln Pro Asp Leu Trp Lys Phe Ala Pro Val Lys 575 580 585 Val Ser Trp Asp 5 671 PRT Homo sapiens misc_feature Incyte ID No 4728247CD1 5 Met Ser Glu Leu Leu Asp Leu Ser Phe Leu Ser Glu Glu Glu Lys 1 5 10 15 Asp Leu Ile Leu Ser Val Leu Gln Arg Asp Glu Glu Val Arg Lys 20 25 30 Ala Asp Glu Lys Arg Ile Arg Arg Leu Lys Asn Glu Leu Leu Glu 35 40 45 Ile Lys Arg Lys Gly Ala Lys Arg Gly Ser Gln His Tyr Ser Asp 50 55 60 Arg Thr Cys Ala Arg Cys Gln Glu Ser Leu Gly Arg Leu Ser Pro 65 70 75 Lys Thr Asn Thr Cys Arg Gly Cys Asn His Leu Val Cys Arg Asp 80 85 90 Cys Arg Ile Gln Glu Ser Asn Gly Thr Trp Arg Cys Lys Val Cys 95 100 105 Ala Lys Glu Ile Glu Leu Lys Lys Ala Thr Gly Asp Trp Phe Tyr 110 115 120 Asp Gln Lys Val Asn Arg Phe Ala Tyr Arg Thr Gly Ser Glu Ile 125 130 135 Ile Arg Met Ser Leu Arg His Lys Pro Ala Val Ser Lys Arg Glu 140 145 150 Thr Val Gly Gln Ser Leu Leu His Gln Thr Gln Met Gly Asp Ile 155 160 165 Trp Pro Gly Arg Lys Ile Ile Gln Glu Arg Gln Lys Glu Pro Ser 170 175 180 Val Leu Phe Glu Val Pro Lys Leu Lys Ser Gly Lys Ser Ala Leu 185 190 195 Glu Ala Glu Ser Glu Ser Leu Asp Ser Phe Thr Ala Asp Ser Asp 200 205 210 Ser Thr Ser Arg Arg Asp Ser Leu Asp Lys Ser Gly Leu Phe Pro 215 220 225 Glu Trp Lys Lys Met Ser Ala Pro Lys Ser Gln Val Glu Lys Glu 230 235 240 Thr Gln Pro Gly Gly Gln Asn Val Val Phe Val Asp Glu Gly Glu 245 250 255 Met Ile Phe Lys Lys Asn Thr Arg Lys Ile Leu Arg Pro Ser Glu 260 265 270 Tyr Thr Lys Ser Val Ile Asp Leu Arg Pro Glu Asp Val Val His 275 280 285 Glu Ser Gly Ser Leu Gly Asp Arg Ser Lys Ser Val Pro Gly Leu 290 295 300 Asn Val Asp Met Glu Glu Glu Glu Glu Glu Glu Asp Ile Asp His 305 310 315 Leu Val Lys Leu His Arg Gln Lys Leu Ala Arg Ser Ser Met Gln 320 325 330 Ser Gly Ser Ser Met Ser Thr Ile Gly Ser Met Met Ser Ile Tyr 335 340 345 Ser Glu Ala Gly Asp Phe Gly Asn Ile Phe Val Thr Gly Arg Ile 350 355 360 Ala Phe Ser Leu Lys Tyr Glu Gln Gln Thr Gln Ser Leu Val Val 365 370 375 His Val Lys Glu Cys His Gln Leu Ala Tyr Ala Asp Glu Ala Lys 380 385 390 Lys Arg Ser Asn Pro Tyr Val Lys Thr Tyr Leu Leu Pro Asp Lys 395 400 405 Ser Arg Gln Gly Lys Arg Lys Thr Ser Ile Lys Arg Asp Thr Val 410 415 420 Asn Pro Leu Tyr Asp Glu Thr Leu Arg Tyr Glu Ile Pro Glu Ser 425 430 435 Leu Leu Ala Gln Arg Thr Leu Gln Phe Ser Val Trp His His Gly 440 445 450 Arg Phe Gly Arg Asn Thr Phe Leu Gly Glu Ala Glu Ile Gln Met 455 460 465 Asp Ser Trp Lys Leu Asp Lys Lys Leu Asp His Cys Leu Pro Leu 470 475 480 His Gly Lys Ile Ser Ala Glu Ser Pro Thr Gly Leu Pro Ser His 485 490 495 Lys Gly Glu Leu Val Val Ser Leu Lys Tyr Ile Pro Ala Ser Lys 500 505 510 Thr Pro Val Gly Gly Asp Arg Lys Lys Ser Lys Gly Gly Glu Gly 515 520 525 Gly Glu Leu Gln Val Trp Ile Lys Glu Ala Lys Asn Leu Thr Ala 530 535 540 Ala Lys Ala Gly Gly Thr Ser Asp Ser Phe Val Lys Gly Tyr Leu 545 550 555 Leu Pro Met Arg Asn Lys Ala Ser Lys Arg Lys Thr Pro Val Met 560 565 570 Lys Lys Thr Leu Asn Pro His Tyr Asn His Thr Phe Val Tyr Asn 575 580 585 Gly Val Arg Leu Glu Asp Leu Gln His Met Cys Leu Glu Leu Thr 590 595 600 Val Trp Asp Arg Glu Pro Leu Ala Ser Asn Asp Phe Leu Gly Gly 605 610 615 Val Arg Leu Gly Val Gly Thr Gly Ile Ser Asn Gly Glu Val Val 620 625 630 Asp Trp Met Asp Ser Thr Gly Glu Glu Val Ser Leu Trp Gln Lys 635 640 645 Met Arg Gln Tyr Pro Gly Ser Trp Ala Glu Gly Thr Leu Gln Leu 650 655 660 Arg Ser Ser Met Ala Lys Gln Lys Leu Gly Leu 665 670 6 1519 PRT Homo sapiens misc_feature Incyte ID No 7472259CD1 6 Met His Arg Glu Arg Asp Gly Val Val Arg Gln Ala Arg Glu Leu 1 5 10 15 Gln Arg Gln Leu Ala Glu Glu Leu Val Asn Arg Gly His Cys Ser 20 25 30 Arg Pro Gly Ala Ser Glu Val Ser Ala Ala Gln Cys Arg Cys Arg 35 40 45 Leu Gln Glu Val Leu Ala Gln Leu Arg Trp Gln Thr Asp Gly Glu 50 55 60 Gln Ala Ala Arg Ile Arg Tyr Leu Gln Ala Ala Leu Glu Val Glu 65 70 75 Arg Gln Leu Phe Leu Lys Tyr Ile Leu Ala His Phe Arg Gly His 80 85 90 Pro Ala Leu Ser Gly Ser Pro Asp Pro Gln Ala Val His Ser Leu 95 100 105 Glu Glu Pro Leu Pro Gln Thr Ser Ser Gly Ser Cys His Ala Pro 110 115 120 Lys Pro Ala Cys Gln Leu Gly Ser Leu Asp Ser Leu Ser Ala Glu 125 130 135 Val Gly Val Arg Ser Arg Ser Leu Gly Leu Val Ser Ser Ala Cys 140 145 150 Ser Ser Ser Pro Asp Gly Leu Leu Ser Thr His Ala Ser Ser Leu 155 160 165 Asp Cys Phe Ala Pro Ala Cys Ser Arg Ser Leu Asp Ser Thr Arg 170 175 180 Ser Leu Pro Lys Ala Ser Lys Ser Glu Glu Arg Pro Ser Ser Pro 185 190 195 Asp Thr Ser Thr Pro Gly Ser Arg Arg Leu Ser Pro Pro Pro Ser 200 205 210 Pro Leu Pro Pro Pro Pro Pro Pro Ser Ala His Arg Lys Leu Ser 215 220 225 Asn Pro Arg Gly Gly Glu Gly Ser Glu Ser Gln Pro Cys Glu Val 230 235 240 Leu Thr Pro Ser Pro Pro Gly Leu Gly His His Glu Leu Ile Lys 245 250 255 Leu Asn Trp Leu Leu Ala Lys Ala Leu Trp Val Leu Ala Arg Arg 260 265 270 Cys Tyr Thr Leu Gln Glu Glu Asn Lys Gln Leu Arg Arg Ala Gly 275 280 285 Cys Pro Tyr Gln Ala Asp Glu Lys Val Lys Arg Leu Lys Val Lys 290 295 300 Arg Ala Glu Leu Thr Gly Leu Ala Arg Arg Leu Ala Asp Arg Ala 305 310 315 Arg Glu Leu Gln Glu Thr Asn Leu Arg Ala Val Ser Ala Pro Ile 320 325 330 Pro Gly Glu Ser Cys Ala Gly Leu Glu Leu Cys Gln Val Phe Ala 335 340 345 Arg Gln Arg Ala Arg Asp Leu Ser Glu Gln Ala Ser Ala Pro Leu 350 355 360 Ala Lys Asp Lys Gln Ile Glu Glu Leu Arg Gln Glu Cys His Leu 365 370 375 Leu Gln Ala Arg Val Ala Ser Gly Pro Cys Ser Asp Leu His Thr 380 385 390 Gly Arg Gly Gly Pro Cys Thr Gln Trp Leu Asn Val Arg Asp Leu 395 400 405 Asp Arg Leu Gln Arg Glu Ser Gln Arg Glu Val Leu Arg Leu Gln 410 415 420 Arg Gln Leu Met Leu Gln Gln Gly Asn Gly Gly Ala Trp Pro Glu 425 430 435 Ala Gly Gly Gln Ser Ala Thr Cys Glu Glu Val Arg Arg Gln Met 440 445 450 Leu Ala Leu Glu Arg Glu Leu Asp Gln Arg Arg Arg Glu Cys Gln 455 460 465 Glu Leu Gly Ala Gln Ala Ala Pro Ala Arg Arg Arg Gly Glu Glu 470 475 480 Ala Glu Thr Gln Leu Gln Ala Ala Leu Leu Lys Asn Ala Trp Leu 485 490 495 Ala Glu Glu Asn Gly Arg Leu Gln Ala Lys Thr Asp Trp Val Arg 500 505 510 Lys Val Glu Ala Glu Asn Ser Glu Val Arg Gly His Leu Gly Arg 515 520 525 Ala Cys Gln Glu Arg Asp Ala Ser Gly Leu Ile Ala Glu Gln Leu 530 535 540 Leu Gln Gln Ala Ala Arg Gly Gln Asp Arg Gln Gln Gln Leu Gln 545 550 555 Arg Asp Pro Gln Lys Ala Leu Cys Asp Leu His Pro Ser Trp Lys 560 565 570 Glu Ile Gln Ala Leu Gln Cys Arg Pro Gly His Pro Pro Glu Gln 575 580 585 Pro Trp Glu Thr Ser Gln Met Pro Glu Ser Gln Val Lys Gly Ser 590 595 600 Arg Arg Pro Lys Phe His Ala Arg Pro Glu Asp Tyr Ala Val Ser 605 610 615 Gln Pro Asn Arg Asp Ile Gln Glu Lys Arg Glu Ala Ser Leu Glu 620 625 630 Glu Ser Pro Val Ala Leu Gly Glu Ser Ala Ser Val Pro Gln Val 635 640 645 Ser Glu Thr Val Pro Ala Ser Gln Pro Leu Ser Lys Lys Thr Ser 650 655 660 Ser Gln Ser Asn Ser Ser Ser Glu Gly Ser Met Trp Ala Thr Val 665 670 675 Pro Ser Ser Pro Thr Leu Asp Arg Asp Thr Ala Ser Glu Val Asp 680 685 690 Asp Leu Glu Pro Asp Ser Val Ser Leu Ala Leu Glu Met Gly Gly 695 700 705 Ser Ala Ala Pro Ala Ala Pro Lys Leu Lys Ile Phe Met Ala Gln 710 715 720 Tyr Asn Tyr Asn Pro Phe Glu Gly Pro Asn Asp His Pro Glu Gly 725 730 735 Glu Leu Pro Leu Thr Ala Gly Asp Tyr Ile Tyr Ile Phe Gly Asp 740 745 750 Met Asp Glu Asp Gly Phe Tyr Glu Gly Glu Leu Asp Asp Gly Arg 755 760 765 Arg Gly Leu Val Pro Ser Asn Phe Val Glu Gln Ile Pro Asp Ser 770 775 780 Tyr Ile Pro Gly Cys Leu Pro Ala Lys Ser Pro Asp Leu Gly Pro 785 790 795 Ser Gln Leu Pro Ala Gly Gln Asp Glu Ala Leu Glu Glu Asp Ser 800 805 810 Leu Leu Ser Gly Lys Ala Gln Gly Met Val Asp Arg Gly Leu Cys 815 820 825 Gln Met Val Arg Val Gly Ser Lys Thr Glu Val Ala Thr Glu Ile 830 835 840 Leu Asp Thr Lys Thr Glu Ala Cys Gln Leu Gly Leu Leu Gln Ser 845 850 855 Met Gly Lys Gln Gly Leu Ser Arg Pro Leu Leu Gly Thr Lys Gly 860 865 870 Val Leu Arg Met Ala Pro Met Gln Leu His Leu Gln Asn Val Thr 875 880 885 Ala Thr Ser Ala Asn Ile Thr Trp Val Tyr Ser Ser His Arg His 890 895 900 Pro His Val Val Tyr Leu Asp Asp Arg Glu His Ala Leu Thr Pro 905 910 915 Ala Gly Val Ser Cys Tyr Thr Phe Gln Gly Leu Cys Pro Gly Thr 920 925 930 His Tyr Arg Val Arg Val Glu Val Arg Leu Pro Trp Asp Leu Leu 935 940 945 Gln Val Tyr Trp Gly Thr Met Ser Ser Thr Val Thr Phe Asp Thr 950 955 960 Leu Leu Ala Gly Pro Pro Tyr Pro Pro Leu Asp Val Leu Val Glu 965 970 975 Arg His Ala Ser Pro Gly Val Leu Val Val Ser Trp Leu Pro Val 980 985 990 Thr Ile Asp Ser Ala Gly Ser Ser Asn Gly Val Gln Val Thr Gly 995 1000 1005 Tyr Ala Val Tyr Ala Asp Gly Leu Lys Val Cys Glu Val Ala Asp 1010 1015 1020 Ala Thr Ala Gly Ser Thr Val Leu Glu Phe Ser Gln Leu Gln Val 1025 1030 1035 Pro Leu Thr Trp Gln Lys Val Ser Val Arg Thr Met Ser Leu Cys 1040 1045 1050 Gly Glu Ser Leu Asp Ser Val Pro Ala Gln Ile Pro Glu Asp Phe 1055 1060 1065 Phe Met Cys His Arg Trp Pro Glu Thr Pro Pro Phe Ser Tyr Thr 1070 1075 1080 Cys Gly Asp Pro Ser Thr Tyr Arg Val Thr Phe Pro Val Cys Pro 1085 1090 1095 Gln Lys Leu Ser Leu Ala Pro Pro Ser Ala Lys Ala Ser Pro His 1100 1105 1110 Asn Pro Gly Ser Cys Gly Glu Pro Gln Ala Lys Phe Leu Glu Ala 1115 1120 1125 Phe Phe Glu Glu Pro Pro Arg Arg Gln Ser Pro Val Ser Asn Leu 1130 1135 1140 Gly Ser Glu Gly Glu Cys Pro Ser Ser Gly Ala Gly Ser Gln Ala 1145 1150 1155 Gln Glu Leu Ala Glu Ala Trp Glu Gly Cys Arg Lys Asp Leu Leu 1160 1165 1170 Phe Gln Lys Ser Pro Gln Asn His Arg Pro Pro Ser Val Ser Asp 1175 1180 1185 Gln Pro Gly Glu Lys Glu Asn Cys Tyr Gln His Met Gly Thr Ser 1190 1195 1200 Lys Ser Pro Ala Pro Gly Phe Ile His Leu Arg Thr Glu Cys Gly 1205 1210 1215 Pro Arg Lys Glu Pro Cys Gln Glu Lys Ala Ala Leu Glu Arg Val 1220 1225 1230 Leu Arg Gln Lys Gln Asp Ala Gln Gly Phe Thr Pro Pro Gln Leu 1235 1240 1245 Gly Ala Ser Gln Gln Tyr Ala Ser Asp Phe His Asn Val Leu Lys 1250 1255 1260 Glu Glu Gln Glu Ala Leu Cys Leu Asp Leu Trp Gly Thr Glu Arg 1265 1270 1275 Arg Glu Glu Arg Arg Glu Pro Glu Pro His Ser Arg Gln Gly Gln 1280 1285 1290 Ala Leu Gly Val Lys Arg Gly Cys Gln Leu His Glu Pro Ser Ser 1295 1300 1305 Ala Leu Cys Pro Ala Pro Ser Ala Lys Val Ile Lys Met Pro Arg 1310 1315 1320 Gly Gly Pro Gln Gln Leu Gly Thr Gly Ala Asn Thr Pro Ala Arg 1325 1330 1335 Val Phe Val Ala Leu Ser Asp Tyr Asn Pro Leu Val Met Ser Ala 1340 1345 1350 Asn Leu Lys Ala Ala Glu Glu Glu Leu Val Phe Gln Lys Arg Gln 1355 1360 1365 Leu Leu Arg Val Trp Gly Ser Gln Asp Thr His Asp Phe Tyr Leu 1370 1375 1380 Ser Glu Cys Asn Arg Gln Val Gly Asn Ile Pro Gly Arg Leu Val 1385 1390 1395 Ala Glu Met Glu Val Gly Thr Glu Gln Thr Asp Arg Arg Trp Arg 1400 1405 1410 Ser Pro Ala Gln Gly His Leu Pro Ser Val Ala His Leu Glu Asp 1415 1420 1425 Phe Gln Gly Leu Thr Ile Pro Gln Gly Ser Ser Leu Val Leu Gln 1430 1435 1440 Gly Asn Ser Lys Arg Leu Pro Leu Trp Thr Pro Lys Ile Met Ile 1445 1450 1455 Ala Ala Leu Asp Tyr Asp Pro Gly Asp Gly Gln Met Gly Gly Gln 1460 1465 1470 Gly Lys Gly Arg Leu Ala Leu Arg Ala Gly Asp Val Val Met Val 1475 1480 1485 Tyr Gly Pro Met Asp Asp Gln Gly Phe Tyr Tyr Gly Glu Leu Gly 1490 1495 1500 Gly His Arg Gly Leu Val Pro Ala His Leu Leu Asp His Met Ser 1505 1510 1515 Leu His Gly His 7 396 PRT Homo sapiens misc_feature Incyte ID No 7476740CD1 7 Met Leu Ile Thr Val Tyr Cys Val Arg Arg Asp Leu Ser Glu Val 1 5 10 15 Thr Phe Ser Leu Gln Val Ser Pro Asp Phe Glu Leu Arg Asn Phe 20 25 30 Lys Val Leu Cys Glu Ala Glu Ser Arg Val Pro Val Glu Glu Ile 35 40 45 Gln Ile Ile His Met Glu Arg Leu Leu Ile Glu Asp His Cys Ser 50 55 60 Leu Gly Ser Tyr Gly Leu Lys Asp Gly Asp Ile Val Val Leu Leu 65 70 75 Gln Lys Asp Asn Val Gly Pro Arg Ala Pro Gly Arg Ala Pro Asn 80 85 90 Gln Pro Arg Val Asp Phe Ser Gly Ile Ala Val Pro Gly Thr Ser 95 100 105 Ser Ser Arg Pro Gln His Pro Gly Gln Gln Gln Gln Arg Thr Pro 110 115 120 Ala Ala Gln Arg Ser Gln Gly Leu Ala Ser Gly Glu Lys Val Ala 125 130 135 Gly Leu Gln Gly Leu Gly Ser Pro Ala Leu Ile Arg Ser Met Leu 140 145 150 Leu Ser Asn Pro His Asp Leu Ser Leu Leu Lys Glu Arg Asn Pro 155 160 165 Pro Leu Ala Glu Ala Leu Leu Ser Gly Ser Leu Glu Thr Phe Ser 170 175 180 Gln Val Leu Met Glu Gln Gln Arg Glu Lys Ala Leu Arg Glu Gln 185 190 195 Glu Arg Leu Arg Leu Tyr Thr Ala Asp Pro Leu Asp Arg Glu Ala 200 205 210 Gln Ala Lys Ile Glu Glu Glu Ile Arg Gln Gln Asn Ile Glu Glu 215 220 225 Asn Met Asn Ile Ala Ile Glu Glu Ala Pro Glu Ser Phe Gly Gln 230 235 240 Val Thr Met Leu Tyr Ile Asn Cys Lys Val Asn Gly His Pro Leu 245 250 255 Lys Ala Phe Val Asp Ser Gly Ala Gln Met Thr Ile Met Ser Gln 260 265 270 Ala Cys Ala Glu Arg Cys Asn Ile Met Arg Leu Val Asp Arg Arg 275 280 285 Trp Ala Gly Val Ala Lys Gly Val Gly Thr Gln Arg Ile Ile Gly 290 295 300 Arg Val His Leu Ala Gln Ile Gln Ile Glu Gly Asp Phe Leu Gln 305 310 315 Cys Ser Phe Ser Ile Leu Glu Asp Gln Pro Met Asp Met Leu Leu 320 325 330 Gly Leu Asp Met Leu Arg Arg His Gln Cys Ser Ile Asp Leu Lys 335 340 345 Lys Asn Val Leu Val Ile Gly Thr Thr Gly Thr Gln Thr Tyr Phe 350 355 360 Leu Pro Glu Gly Glu Leu Pro Leu Cys Ser Arg Met Val Ser Gly 365 370 375 Gln Asp Glu Ser Ser Asp Lys Glu Ile Thr His Ser Val Met Asp 380 385 390 Ser Gly Arg Lys Glu His 395 8 590 PRT Homo sapiens misc_feature Incyte ID No 7473774CD1 8 Met Ser Gly Asp Tyr Glu Asp Asp Leu Cys Arg Arg Ala Leu Ile 1 5 10 15 Leu Val Ser Asp Leu Cys Ala Arg Val Arg Asp Ala Asp Thr Asn 20 25 30 Asp Arg Cys Gln Glu Phe Asn Asp Arg Ile Arg Gly Tyr Pro Arg 35 40 45 Gly Pro Asp Ala Asp Ile Ser Val Ser Leu Leu Ser Val Ile Val 50 55 60 Thr Phe Cys Gly Ile Val Leu Leu Gly Val Ser Leu Phe Val Ser 65 70 75 Trp Lys Leu Cys Trp Val Pro Trp Arg Asp Lys Gly Gly Ser Ala 80 85 90 Val Gly Gly Gly Pro Leu Arg Lys Asp Leu Gly Pro Gly Val Gly 95 100 105 Leu Ala Gly Leu Val Gly Gly Gly Gly His His Leu Ala Ala Gly 110 115 120 Leu Gly Gly His Pro Leu Leu Gly Gly Pro His His His Ala His 125 130 135 Ala Ala His His Pro Pro Phe Ala Glu Leu Leu Glu Pro Gly Ser 140 145 150 Leu Gly Gly Ser Asp Thr Pro Glu Pro Ser Tyr Leu Asp Met Asp 155 160 165 Ser Tyr Pro Glu Ala Ala Ala Ala Ala Val Ala Ala Gly Val Lys 170 175 180 Pro Ser Gln Thr Ser Pro Glu Leu Pro Ser Glu Gly Gly Ala Gly 185 190 195 Ser Gly Leu Leu Leu Leu Pro Pro Ser Gly Gly Gly Leu Pro Ser 200 205 210 Ala Gln Ser His Gln Gln Val Thr Ser Leu Ala Pro Thr Thr Arg 215 220 225 Tyr Pro Ala Leu Pro Arg Pro Leu Thr Gln Gln Thr Leu Thr Ser 230 235 240 Gln Pro Asp Pro Ser Ser Glu Glu Arg Pro Pro Ala Leu Pro Leu 245 250 255 Pro Leu Pro Gly Gly Glu Glu Lys Ala Lys Leu Ile Gly Gln Ile 260 265 270 Lys Pro Glu Leu Tyr Gln Gly Thr Gly Pro Gly Gly Arg Arg Ser 275 280 285 Gly Gly Gly Pro Gly Ser Gly Glu Ala Gly Thr Gly Ala Pro Cys 290 295 300 Gly Arg Ile Ser Phe Ala Leu Arg Tyr Leu Tyr Gly Ser Asp Gln 305 310 315 Leu Val Val Arg Ile Leu Gln Ala Leu Asp Leu Pro Ala Lys Asp 320 325 330 Ser Asn Gly Phe Ser Asp Pro Tyr Val Lys Ile Tyr Leu Leu Pro 335 340 345 Asp Arg Lys Lys Lys Phe Gln Thr Lys Val His Arg Lys Thr Leu 350 355 360 Asn Pro Val Phe Asn Glu Thr Phe Gln Phe Ser Val Pro Leu Ala 365 370 375 Glu Leu Ala Gln Arg Lys Leu His Phe Ser Val Tyr Asp Phe Asp 380 385 390 Arg Phe Ser Arg His Asp Leu Ile Gly Gln Val Val Leu Asp Asn 395 400 405 Leu Leu Glu Leu Ala Glu Gln Pro Pro Asp Arg Pro Leu Trp Arg 410 415 420 Asp Ile Val Glu Gly Gly Ser Glu Lys Ala Asp Leu Gly Glu Leu 425 430 435 Asn Phe Ser Leu Cys Tyr Leu Pro Thr Ala Gly Arg Leu Thr Val 440 445 450 Thr Ile Ile Lys Ala Ser Asn Leu Lys Ala Met Asp Leu Thr Gly 455 460 465 Phe Ser Asp Pro Tyr Val Lys Ala Ser Leu Ile Ser Glu Gly Arg 470 475 480 Arg Leu Lys Lys Arg Lys Thr Ser Ile Lys Lys Asn Thr Leu Asn 485 490 495 Pro Thr Tyr Asn Glu Ala Leu Val Phe Asp Val Ala Pro Glu Ser 500 505 510 Val Glu Asn Val Gly Leu Ser Ile Ala Val Val Asp Tyr Asp Cys 515 520 525 Ile Gly His Asn Glu Val Ile Gly Val Cys Arg Val Gly Pro Asp 530 535 540 Ala Ala Asp Pro His Gly Arg Glu His Trp Ala Glu Met Leu Ala 545 550 555 Asn Pro Arg Lys Pro Val Glu His Trp His Gln Leu Val Glu Glu 560 565 570 Lys Thr Val Thr Ser Phe Thr Lys Gly Ser Lys Gly Leu Ser Glu 575 580 585 Lys Glu Asn Ser Glu 590 9 431 PRT Homo sapiens misc_feature Incyte ID No 7946329CD1 9 Met Ala Glu Ile Thr Asn Ile Arg Pro Ser Phe Asp Val Ser Pro 1 5 10 15 Val Val Ala Gly Leu Ile Gly Ala Ser Val Leu Val Val Cys Val 20 25 30 Ser Val Thr Val Phe Val Trp Ser Cys Cys His Gln Gln Ala Glu 35 40 45 Lys Lys His Lys Asn Pro Pro Tyr Lys Phe Ile His Met Leu Lys 50 55 60 Gly Ile Ser Ile Tyr Pro Glu Thr Leu Ser Asn Lys Lys Lys Ile 65 70 75 Ile Lys Val Arg Arg Asp Lys Asp Gly Pro Gly Arg Glu Gly Gly 80 85 90 Arg Arg Asn Leu Leu Val Asp Ala Ala Glu Ala Gly Leu Leu Ser 95 100 105 Arg Asp Lys Asp Pro Arg Gly Pro Ser Ser Gly Ser Cys Ile Asp 110 115 120 Gln Leu Pro Ile Lys Met Asp Tyr Gly Glu Glu Leu Arg Ser Pro 125 130 135 Ile Thr Ser Leu Thr Pro Gly Glu Ser Lys Thr Thr Ser Pro Ser 140 145 150 Ser Pro Glu Glu Asp Val Met Leu Gly Ser Leu Thr Phe Ser Val 155 160 165 Asp Tyr Asn Phe Pro Lys Lys Ala Leu Val Val Thr Ile Gln Glu 170 175 180 Ala His Gly Leu Pro Val Met Asp Asp Gln Thr Gln Gly Ser Asp 185 190 195 Pro Tyr Ile Lys Met Thr Ile Leu Pro Asp Lys Arg His Arg Val 200 205 210 Lys Thr Arg Val Leu Arg Lys Thr Leu Asp Pro Val Phe Asp Glu 215 220 225 Thr Phe Thr Phe Tyr Gly Ile Pro Tyr Ser Gln Leu Gln Asp Leu 230 235 240 Val Leu His Phe Leu Val Leu Ser Phe Asp Arg Phe Ser Arg Asp 245 250 255 Asp Val Ile Gly Glu Val Met Val Pro Leu Ala Gly Val Asp Pro 260 265 270 Ser Thr Gly Lys Val Gln Leu Thr Arg Asp Ile Ile Lys Arg Asn 275 280 285 Ile Gln Lys Cys Ile Ser Arg Gly Glu Leu Gln Val Ser Leu Ser 290 295 300 Tyr Gln Pro Val Ala Gln Arg Met Thr Val Val Val Leu Lys Ala 305 310 315 Arg His Leu Pro Lys Met Asp Ile Thr Gly Leu Ser Gly Asn Pro 320 325 330 Tyr Val Lys Val Asn Val Tyr Tyr Gly Arg Lys Arg Ile Ala Lys 335 340 345 Lys Lys Thr His Val Lys Lys Cys Thr Leu Asn Pro Ile Phe Asn 350 355 360 Glu Ser Phe Ile Tyr Asp Ile Pro Thr Asp Leu Leu Pro Asp Ile 365 370 375 Ser Ile Glu Phe Leu Val Ile Asp Phe Asp Arg Thr Thr Lys Asn 380 385 390 Glu Val Val Gly Arg Leu Ile Leu Gly Ala His Ser Val Thr Ala 395 400 405 Ser Gly Ala Glu His Trp Arg Glu Val Cys Glu Ser Pro Arg Lys 410 415 420 Pro Val Ala Lys Trp His Ser Leu Ser Glu Tyr 425 430 10 3424 DNA Homo sapiens misc_feature Incyte ID No 1577952CB1 10 cgcacgtgcg cgcgaagacg tggggacgca ggcgggtcgt agagagcgtt cagccgtctg 60 tatatctccc cagatacctg aaactgacca cctgagtacg ttttcccatt gctgagctgt 120 ttccctgata tctggccatg caacggagat caagagggat aaatactgga cttattctac 180 tcctttctca aatcttccat gttgggatca acaatattcc acctgtcacc ctagcaactt 240 tggccctcaa catctggttc ttcttgaacc ctcagaagcc actgtatagc tcctgcctta 300 gtgtggagaa gtgttaccag caaaaagact ggcagcgttt actgctctct ccccttcacc 360 atgctgatga ttggcatttg tatttcaata tggcatccat gctctggaaa ggaataaatc 420 tagaaagaag actgggaagt agatggtttg cctatgttat caccgcattt tctgtactta 480 ctggagtggt atacctgctc ttgcaatttg ctgttgccga atttatggat gaacctgact 540 tcaaaaggag ctgtgctgta ggtttctcag gagttttgtt tgctttgaaa gttcttaaca 600 accattattg ccctggaggc tttgtcaaca ttttgggctt tcctgtaccg aacagatttg 660 cttgttgggt cgaacttgtg gctattcatt tattctcacc agggacttcc ttcgctgggc 720 atctggctgg gattcttgtt ggactaatgt acactcaagg gcctctgaag aaaatcatgg 780 aagcatgtgc aggcggtttt tcctccagtg ttggttaccc aggacggcaa tactacttta 840 atagttcagg cagctctgga tatcaggatt attatccgca tggcaggcca gatcactatg 900 aagaagcacc caggaactat gacacgtaca cagcaggact gagtgaagaa gaacagctcg 960 agagagcatt acaagccagc ctctgggacc gaggaaatac cagaaatagc ccaccaccct 1020 acgggtttca tctctcacca gaagaaatga ggagacagcg gcttcacaga ttcgatagcc 1080 agtgaggtgg catcttggga agacatggcc tattcgtgta attattgccc atttggctca 1140 ttccccaagc ccctaattca ttttaattca ttttaaacaa aagcagagta caccggtatt 1200 gctccagatc gctcacatca cctgggacag tcccatggcc cctatgagtc aactcacagc 1260 ttgcggggag tgggccttct cctggccttg ttcttgctca taaacaggtc acttcctcca 1320 tgaagagacc agtttccacg ctcccatctc tcactgctga ctcagcgatg cctctgcctc 1380 ggtctgcttt tgaagactgt gaccttcacc aggaggtttt acttacacca gtcgggaaga 1440 ttagtccctc attctgcctg gagtgccccg tgtttgactt ggcagcgggt gtggagccat 1500 ccccgcgtcc tcctggcaca ttgccactgt ggctgtccag gaacaggatg tggctgcctt 1560 ggccgaatgt tgtcctactc tccccaaccc cggcgcctca gctcctcagc tcctcgggcc 1620 cctgcgtctg gctggtgttt gcagggcttt cgctctgctc tggtattgct ctgcctttat 1680 agaaagtctt attgaagaag tgtaagaaag acctaaggtg gggaagactc ctacacacac 1740 cattagtatc agtgacacca gcaatgtagg ttcccagccc cttcccagtg gcagcttgtg 1800 tgtccaggag ataggacatc atttaacgca tcagcaaagt agcagcagat gccacataca 1860 gagtagagcg aaggcatttg gtggatcggt cactagagat ctatcttgca gaaagtatgt 1920 ttttcctcat aaaagtgcct cttaattggc cattgtacca gccacttgtc ctagccaaat 1980 gtccaaaaca cgcccttggg ccccgccacg ttacaatcca cagattgtct gtctgagtcg 2040 tttaaggcat ttcctggtgc ttgtgttcca tgaataaaag gacaaagtca gaagatcact 2100 gatgtcttac tgtcaacaga gatattttaa aagagagaag caggaaaaga tcttcctttt 2160 ttgatctaca acttatatag ttttctgatt atgcacataa tagatatgcc ttccagatgc 2220 ataaggcaaa catctggaaa gaaatatacc caaatcttag caggggttat ctttgggagt 2280 ggagtacatg ggattttgct ttcttcattt ttataatttt atattactgt cttggaagat 2340 gtgtttatgt gtgtgtgtta cttttacaat caggaaaaca tatttaataa catatagtca 2400 agaaaacaga cttaaaaata aatactatgt gtccattgag aaaattcaca atataaacag 2460 aaatacaaat aaatacatac acaattttaa agtcacctgt agccctaccc ttagaggtac 2520 ccagggttaa cattttggtg gtattgtctt atcaattttt ccgttgatac attcagcaaa 2580 tttggagcac attgaccatg gagttttgtg tccaaatcca atctgaattt acctggaaga 2640 ggccttgaca cctgcatgga aatgagctaa gaaaaccact ggagccttgg gagctctttg 2700 gcctcctggc tggcccagta atatctgagc tcctttggtt aatttataac tgatataaaa 2760 ctacatcttc tttataatat aaattgtacc tgtgagtcta gaagctttaa atgtgtttaa 2820 attaaaatat tcaagctaaa tgttactgct ctctcccaaa ttctgtaagt ttgactcccg 2880 ttaccccaat tagaagtaac ttctttgttt catgccactt ttatagcatt tggtaattct 2940 gctataacac atcttgcccc tattattaac tgtgcacagc tacacaaagg tgtgccttct 3000 acgtgggaac atggattgtg aatgactctg taatgaggcc tgagtcttag ttatctttcc 3060 actcactccc cgtctcccct ttccaacccc aaaggctcac gataggggct cactaaatgt 3120 cagtgtttca ccaaagtatt ttttccattg tattaagagt ccagtcactg tatatggaag 3180 tattttattt tttatttttt tatatcactt gagtccacta gtagtacttc cttgctctgt 3240 ttgacttgtc agatacaaag acacgggatt agattttggg tggtaaaatt gtgatacgca 3300 tggctgttga tggagtggaa catcttagtg atgtgagaaa ggtcatttta gttataaatg 3360 taaaccaatt actttagcac aacaataaag atgttctgga aattaaaaaa aaaaaaaaaa 3420 aagg 3424 11 1033 DNA Homo sapiens misc_feature Incyte ID No 4983705CB1 11 agcggccgca gcctctgaga gcacgaacag cagcgccccc gcgtcccagc cagccagcca 60 gccagactgg actccggccc accgacggcc gctcgcgctc cggccccgct cgcctgctct 120 gccccggacc tgcagctccc cgctcccccg ccgtgtccgc cgcctcccgg ccagagagcc 180 aagcccccac gccgcgccca gcgctcgccg cgccagcatg tcctcgaccg agagcgccgg 240 ccgcacggcg gacaagtcgc cgcgccagca ggtggaccgc ctactcgtgg ggctgcgctg 300 gcggcggctg gaggagccgc tgggcttcat caaagttctc cagtggctct ttgctatttt 360 cgccttcggg tcctgtggct cctacagcgg ggagacagga gcaatggttc gctgcaacaa 420 cgaagccaag gacgtgagct ccatcatcgt tgcatttggc tatcccttca ggttgcaccg 480 gatccaatat gagatgcccc tctgcgatga agagtccagc tccaagacca tgcacctcat 540 gggggacttc tctgcacccg ccgagttctt cgtgaccctt ggcatctttt ccttcttcta 600 taccatggct gccctagtta tctacctgcg cttccacaac ctctacacag agaacaaacg 660 cttcccgctg gtggacttct gtgtgactgt ctccttcacc ttcttctggc tggtagctgc 720 agctgcctgg ggcaagggcc tgaccgatgt caagggggcc acacgaccat ccagcttgac 780 agcagccatg tcagtgtgcc atggagagga agcagtgtgc agtgccgggg ccacgccctc 840 tatgggcctg gccaacatct ccgtgctctt tggctttatc aacttcttcc tgtgggccgg 900 gaactgttgg tttgtgttca aggagacccc gtggcatgga cagggccagg gccaggacca 960 ggaccaggac caggaccagg gccagggtcc cagccaggag agtgcagctg agcagggagc 1020 agtggagaag cag 1033 12 3902 DNA Homo sapiens misc_feature Incyte ID No 1310465CB1 12 ctttccatca caggccgcac tgctccctct ggcccaacca tgcctctgtc cagccacctg 60 ctgcccgcct tggtcctgtt cctggcagca gggtcctcag gctgggcctg ggtccccaac 120 cactgcagga gccctggcca ggccgtgtgc aacttcgtgt gtgactgcag ggactgctca 180 gatgaggccc agtgtggtta ccacggggcc tcgcccaccc tgggcgcccc cttcgcctgt 240 gacttcgagc aggacccctg cggctggcgg gacattagta cctcaggcta cagctggctc 300 cgagacaggg caggggccgc actggagggt cctgggcctc actcagacca cacactgggc 360 accgacttgg gctggtacat ggccgttgga acccaccgag ggaaagaggc atccaccgca 420 gccctgcgct cgccaaccct gcgagaggca gcctcctctt gcaagctgag gctctggtac 480 cacgcggcct ctggagatgt ggctgaactg cgggtggagc tgacccatgg cgcagagacc 540 ctgaccctgt ggcagagcac agggccctgg ggccctggct ggcaggagtt ggcagtgacc 600 acaggccgca tccggggtga cttccgagtg accttctctg ccacccgaaa tgccacccac 660 aggggcgctg tggctctaga tgacctagag ttctgggact gtggtctgcc caccccccag 720 gccaactgtc ccccgggaca ccaccactgc cagaacaagg tctgcgtgga gccccagcag 780 ctgtgcgacg gggaagacaa ctgcggggac ctgtctgatg agaacccact cacctgtggc 840 cgccacatag ccaccgactt tgagacaggc ctgggcccat ggaaccgctc ggaaggctgg 900 tcccggaacc accgtgctgg tggtcctgag cgcccctcct ggccacgccg tgaccacagc 960 cggaacagtg cacagggctc cttcctggtc tccgtggccg agcctggcac ccctgctata 1020 ctctccagcc ccgaattcca agcctcaggc acctccaact gctcgctggt cttctatcag 1080 tacctgagtg ggtctgaggc tggctgcctc cagctgttcc tgcagactct ggggcccggc 1140 gccccccggg cccccgtcct gctgcggagg cgccgagggg agctggggac cgcctgggtc 1200 cgagaccgtg ttgacatcca gagcgcctac cccttccaga tcctcctggc cgggcagaca 1260 ggcccggggg gcgtcgtggg tctggacgac ctcatcctgt ctgaccactg cagaccagtc 1320 tcggaggtgt ccaccctgca gccgctgcct cctgggcccc gggccccagc cccccagccc 1380 ctgccgccca gctcgcggct ccaggattcc tgcaagcagg ggcatcttgc ctgcggggac 1440 ctgtgtgtgc ccccggaaca actgtgtgac ttcgaggagc agtgcgcagg gggcgaggac 1500 gagcaggcct gtggcaccac agactttgag tcccccgagg ctgggggctg ggaggacgcc 1560 agcgtggggc ggctgcagtg gcggcgtgtc tcagcccagg agagccaggg gtccagtgca 1620 gctgctgctg ggcacttcct gtctctgcag cgggcctggg ggcagctagg cgctgaggcc 1680 cgggtcctca cacccctcct tggcccttct ggccccagct gtgaactcca cctggcttat 1740 tatttacaga gccagccccg aggcttcctg gcactagttg tggtggacaa cggctcccgg 1800 gagctggcat ggcaggccct gagcagcagt gcaggcatct ggaaggtgga caaggtcctt 1860 ctaggggccc gccgccggcc cttccggctg gagtttgtcg gtttggtgga cttggatggc 1920 cctgaccagc agggagctgg ggtggacaac gtgaccctga gggactgtag ccccacagtg 1980 accaccgaga gagacagaga ggtctcctgt aactttgagc gggacacatg cagctggtac 2040 ccaggccacc tctcagacac acactggcgc tgggtggaga gccgcggccc tgaccacgac 2100 cacaccacag gccaaggcca ctttgtgctc ctggacccca cagaccccct ggcctggggc 2160 cacagtgccc acctgctctc caggccccag gtgccagcag cacccacgga gtgtctcagc 2220 ttctggtacc acctccatgg gccccagatt gggactctgc gcctagccat gagacgggaa 2280 ggggaggaga cacacctgtg gtcgcggtca ggcacccagg gcaaccgctg gcacgaggcc 2340 tgggccaccc tttcccacca gcctggctcc catgcccagt accagctgct gttcgagggc 2400 ctccgggacg gataccacgg caccatggcg ctggacgatg tggccgtgcg gccgggcccc 2460 tgctgggccc ctaattactg ctcctttgag gactcagact gcggcttctc ccctggaggc 2520 caaggtctct ggaggcggca ggccaatgcc tcgggccatg ctgcctgggg ccccccaaca 2580 gaccatacca ctgagacagc ccaagggcac tacatggtgg tggacacaag cccagacgca 2640 ctaccccggg gccagacggc ctccctgacc tccaaggagc acaggcccct ggcccagcct 2700 gcttgtctga ccttctggta ccacgggagc ctccgcagcc caggcaccct gcgggtctac 2760 ctggaggagc gcgggaggca ccaggtgctc agcctcagtg cccacggcgg gcttgcctgg 2820 cgcctgggca gcatggacgt gcaggccgag cgagcctgga gggtggtgtt tgaggcagtg 2880 gccgcaggcg tggcacactc ctacgtggct ctggatgatc tgctcctcca ggacgggccc 2940 tgccctcagc caggttcctg tgattttgag tctggcctgt gtggctggag ccacctggcc 3000 gggcccggcc tgggcggata cagctgggac tggggcgggg gagccacccc ctctcgttac 3060 ccccagcccc ctgtggacca caccctgggc acagaggcag gccactttgc cttctttgaa 3120 actggcgtgc tgggccccgg gggccgggcc gcctggctgc gcagcgagcc tctgccggcc 3180 accccagcct cctgcctccg cttctggtac cacatgggtt ttcctgagca cttctacaag 3240 ggggagctga aggtactgct gcacagtgct cagggccagc tggctgtgtg gggcgcaggc 3300 gggcatcggc ggcaccagtg gctggaggcc caggtggagg tagccagtgc caaggagttc 3360 cagatcgtgt ttgaagccac tctgggcggc cagccagccc tggggcccat tgccctggat 3420 gacgtggagt atctggctgg gcagcattgc cagcagcctg cccccagccc ggggaacaca 3480 gccgcacccg ggtctgtgcc agctgtggtt ggcagtgccc tcctattgct catgctcctg 3540 gtgctgctgg gacttggggg acggcgctgg ctgcagaaga aggggagctg ccccttccag 3600 agcaacacag aggccacagc ccctggcttt gacaacatcc ttttcaatgc ggatggtgtc 3660 accctcccgg catctgtcac cagtgatccg tagaccaccc cagacaaggc cccgcttcct 3720 cacgtgacat ccagcacttg gtcagaccct agccagggac cggacacctg ccccgcccag 3780 gctgggacag gctgcaggtc tcaggatatg ctgaggcctg ggcgttccct gccctgtgct 3840 gactctgttg ctctgtgaat aaacaccctg gcccatgagg gcagcccaaa aaaaaaaaaa 3900 aa 3902 13 2574 DNA Homo sapiens misc_feature Incyte ID No 4291779CB1 13 cggctcgagg tgcggtcatg gtgggccaga tgtactgcta ccccggcagc cacctggccc 60 gggcgctgac gcgggcgctg gcgctggccc tggtgctggc cctgctggtc gggccgttcc 120 tgagcggcct ggcgggggcg atcccagcgc cggggggccg ctgggcgcgc gatgggccgg 180 tccctccagc ctcccgcagc cgctcggtgc tcctggacgt ctcggcgggc cagctgctta 240 tggtggacgg acgccaccct gacgccgtgg cctgggccaa cctcaccaac gccatccgcg 300 agactgggtg ggccttcctg gagctgggca caagtggcca atacaatgac agcttgcagg 360 cctatgcagc cggtgtggtg gaggctgctg tgtcggagga gctcatctac atgcactgga 420 tgaacacggt ggtgaattac tgcggcccct tcgagtatga agtcggctac tgcgagaggc 480 tgaagagctt cctggaggcc aacctagagt ggatgcagga agagatggag tcaaacccag 540 actcacctta ctggcaccag gtgcggctga ccctcctgca gctgaaaggc ctggaggaca 600 gctacgaagg ccgtgtgagc ttcccagctg ggaagttcac catcaaaccc ttggggttcc 660 tcctgctgca gctctctggg gacctggaag acctggagct ggccctgaac aagaccaaga 720 tcaaaccttc tctgggctct ggctcctgtt ctgccctcat caagctgctc cctggccaga 780 gtgacctcct ggttgcccac aacacctgga acaactacca gcacatgctg cgtgtcatca 840 agaagtactg gctccagttc cgggaaggcc cctgggggga ctacccgctg gttcccggca 900 acaagctggt cttctcctcc taccccggca ccatcttctc ctgcgacgac ttctacatcc 960 tgggcagtgg gctggtgaca ctggagacca ccattggcaa caagaaccca gccctgtgga 1020 agtatgtgcg gcccaggggc tgtgtgctgg agtgggtacg caacatcgtg gccaaccgcc 1080 tggcctcgga tggggccacc tgggcagaca tcttcaagag gttcaacagc ggcacgtata 1140 acaaccagtg gatgatcgtg gactacaagg cgttcatccc gggtgggccc agccccggga 1200 gccgggtgct taccatcctg gagcagatcc ccggcatggt ggtggtggct gacaagacct 1260 cggagctcta ccagaagacc tactgggcca gctacaacat accgtccttc gagactgtgt 1320 tcaatgccag tgggctgcag gccctagtgg cccagtatgg ggactggttt tcttatgacg 1380 ggagcccccg ggcccagatc ttccggcgga accagtcact ggtacaagac atggactcca 1440 tggtcaggct gatgaggtac aatgacttcc tccatgaccc tctgtcactg tgcaaagcct 1500 gcaaccccca gcccaatggg gagaatgcta tctccgcccg ctccgacctc aacccggcca 1560 atggctccta ccccttccag gccctgcgtc agcgctccca tgggggtatc gatgtgaagg 1620 tgaccagcat gtcactggcc aggatcctga gcctgctggc ggccagcggt cccacgtggg 1680 accaggtgcc cccgttccag tggagcacct cgcccttcag cggcctgctg cacatgggcc 1740 agccagacct ctggaagttc gcgcctgtca aggtttcatg ggactgaagt tctgtccctg 1800 ctctgctgct ttcgcccctg ctgaccctcg tcagggtcac ccccgtccca aggccaccgg 1860 acttctaact ccagcccctc ctgggggctt cgttctctga tctggggtct gagtcatctc 1920 ctcctagagt gggtcacgaa cctgatgggg ctcagaactg accccctctc tcccccgagg 1980 tgggtgggca ccgtggcgtc tcttctgccc tgccctaaat ctcccactct ctgtttctgt 2040 ctgtttccta ctgctgctct ctcaacctca ttcccacctc tggggcccct tcctcgtgct 2100 tctccttcct gagggtttgg gaaggtcctg gggcagactc tggggctccc atggggtgga 2160 aggagcctgt tccagcaccc ttctcccagc tgcattccca cgggtggccc tggagctggt 2220 gagctttgtc tgggcgttgt cttcggctgg cattgctcct cccagctctg gcccctctgc 2280 tccctcagga agcagtcccc tcgtctccct ttctgggcag cttccttgag gacagaaact 2340 tgaaaacaaa cacaaaccaa agtttctggc catctgtggc tggagggttc tgaatgtcct 2400 ctctccatgt caggcagagg gtcagccccc atgcttctgc ctcaggcccc accccacccc 2460 accccaggcc tgcccctcac ctcagggcca tacccacagc gccctgatgg aggaaccaga 2520 ccgcaggctg tgccaccatt aaacaagagc ggctgtgaaa aaaaaaaaaa aagg 2574 14 2878 DNA Homo sapiens misc_feature Incyte ID No 4728247CB1 14 gtgaaagagg cgtgttgtct agtttcaaag gagaggagag aaggcaactc tggtagctct 60 ccttgtctgg ttgttttgaa gaaagaagag tagaagaaaa agttgagtaa atcatgtcgg 120 agttactgga cctttctttt ctgtctgagg aggaaaagga tttgattctc agtgttctac 180 agcgagatga agaggtccgg aaagcagatg agaaaaggat taggcgacta aagaatgagt 240 tactggagat aaaaaggaaa ggggccaaga ggggcagcca acactacagt gatcggacct 300 gtgcccggtg ccaggagagc ctgggccgtt tgagtcccaa aaccaatact tgtcggggtt 360 gtaatcacct ggtgtgtcgg gactgccgca tacaggaaag caatggtacc tggaggtgca 420 aggtgtgcgc caaggaaata gagttgaaga aagcaactgg ggactggttt tatgaccaga 480 aagtgaatcg ctttgcttac cgcacaggta gtgagataat caggatgtcc ctgcgccaca 540 aacctgcagt gagtaaaaga gagacagtgg gacagtccct ccttcatcag acacagatgg 600 gtgacatctg gccaggaaga aagatcattc aggagcggca gaaggagccc agtgtgctat 660 ttgaagtgcc aaagctgaaa agtggaaaga gtgcattgga agctgagagt gagagtctgg 720 atagcttcac agctgactcg gatagcacct ccaggagaga ctctctggat aaatctggcc 780 tctttccaga atggaagaag atgtctgctc ccaaatctca agtagaaaag gaaactcagc 840 ctggaggtca aaatgtggta tttgtggatg agggtgagat gatatttaag aagaacacca 900 gaaaaatcct caggccttca gagtacacta aatctgtgat agatcttcgc ccagaagatg 960 tggtacatga aagtggctcc ttgggagaca gaagcaaatc cgtcccaggc ctcaatgtgg 1020 atatggaaga ggaagaagaa gaagaagaca ttgaccacct agtgaagtta catcgccaga 1080 agctagccag aagcagcatg caaagtggct cctccatgag tacgatcggc agcatgatga 1140 gcatctacag tgaagctggt gatttcggga acatctttgt gactggcagg attgcctttt 1200 ccctgaagta tgagcagcaa acccagagtc tggttgtcca tgtgaaggag tgccatcagc 1260 tggcctatgc tgatgaagcc aagaagcgct ctaacccata tgtgaagact taccttctgc 1320 ctgacaagtc ccgccaagga aaaagaaaaa ccagcatcaa gcgggacact gttaatccac 1380 tatatgatga gacgctgagg tatgagatcc cagaatctct cctggcccag aggaccctgc 1440 agttctcagt ttggcatcat ggtcgttttg gcagaaacac tttccttgga gaggcagaga 1500 tccagatgga ttcctggaag cttgataaga aactggatca ttgcctccct ttacatggaa 1560 agatcagtgc tgagtccccg actggcttgc catcacacaa aggcgagttg gtggtttcat 1620 tgaaatacat cccagcctcc aaaacccctg ttggaggtga ccggaaaaag agtaaaggtg 1680 gggaaggggg agagctccag gtgtggatca aagaagccaa gaacttgacg gctgccaaag 1740 caggagggac ttcagacagc tttgtcaagg gatacctcct tcccatgagg aacaaggcca 1800 gtaaacgtaa aactcctgtg atgaagaaga ccctgaatcc tcactacaac catacatttg 1860 tctacaatgg tgtgaggctg gaagatctac agcatatgtg cctggaactg actgtgtggg 1920 accgggagcc cctggccagc aatgacttcc tgggaggggt caggctgggt gttggcactg 1980 ggatcagtaa tggggaagtg gtggactgga tggactcgac tggggaagaa gtgagcctgt 2040 ggcagaagat gcgacagtac ccagggtctt gggcagaagg gactctgcag ctccgttcct 2100 caatggccaa gcagaagctg ggtttatgag tccctgtcct cttctgcagg tccagccctg 2160 gcgagggcag gtcagaggaa gtgaagaaat caagagcaaa gatttataat ttaatgtgta 2220 tgtgtgtatg tgtgtatgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtacaaaca 2280 tgtattttct gcaaatctca ttatgctggc tagagtgatg cagacttgtt cttcttttta 2340 aagcagtctc aagaataagc atttctttaa aatgtttctg tgtataatct agtttatttt 2400 cagagtccat tttttcttat gtctttataa ggttcactta acttaaaaac agcttttaaa 2460 acaacttttt atcttctgtc ttgctatcat tgttcctact tccctaggaa gccctggcta 2520 cctttcgcat taggaccagt ctgggtttta aggctctggg aagcagggtt ggttagtaaa 2580 gacaggaatg ttggggagag gtgagtagtt ccttcctctt tctcctctcc aatttatgct 2640 tttaacttat tttctacctg gataaacttc tggaacttgg cttttaaatt taacttttct 2700 agtttttaag cagtttccac cttgctttgg tctaatgctt ttctttgaaa tgctaacaga 2760 attcccaagc tttttccagt tctagatatc tttactagac ctttggggga ctcttataat 2820 ggagctgctt ttgaaaagca ctttaattag ataatgtatt ttgactaaat cacgagga 2878 15 5628 DNA Homo sapiens misc_feature Incyte ID No 7472259CB1 15 atggccaagg actcgcccag ccccttgggc gcgtcgccca agaagccggg ctgctccagc 60 ccggcggcgg cagtgctgga gaaccagagg cgggagctgg agaagctacg ggcggagctg 120 gaggcggagc gggcaggctg gcgggcggaa cggcggcgct tcgctgcccg ggagcgccag 180 ctgcgtgagg aggccgagcg ggagcggcgg cagctggctg accatctgcg ctccaagtgg 240 gaggcacagc gcagccggga gttgcggcag ctgcaagagg agatgcagcg ggaacgcgag 300 gccgagatcc ggcagctgct gcgctggaac ggaggccgag cagcggcagc tgcagcagct 360 gcatgcaccg ggagcgcgat ggcgtggtgc gccaagcccg ggagctgcag cgccagctgg 420 ccgaggagct ggtgaaccgc ggccactgta gccgcccggg ggcgtccgag gtttccgcgg 480 cgcagtgccg ctgtcgcctg caggaagtgt tggcgcagct tcgctggcag actgacggcg 540 agcaggcggc gcgcatccgc tatctgcagg cggcgctgga ggtggagcgc cagctcttcc 600 tcaagtacat cctggcgcac ttccgcgggc acccggcttt gtcgggatca ccggaccccc 660 aagctgtgca ttccttggaa gaaccgctgc cccagacctc cagcggctct tgccacgccc 720 ccaaacccgc ctgccaactc ggatctctag acagcctgag tgctgaagtc ggtgtgcgct 780 cccgctcgct aggcctggtg tcctctgcgt gctccagctc cccagacggc ctgctctcca 840 cgcacgccag ctcccttgat tgcttcgcac ctgcgtgttc ccgctcgctt gacagcaccc 900 ggagcctccc caaggcctcc aaatccgagg agcggccctc ctcaccagac acctccaccc 960 ctggctcccg gaggctctcg ccgccaccat cgccactccc gccgccacca ccaccgtcag 1020 cccacaggaa actcagcaac ccgcggggag gagaaggctc tgagagccag ccctgcgaag 1080 tcctgactcc ctcacccccg ggcctgggcc accacgagct gataaagctg aactggctgc 1140 tggccaaggc gttgtgggtg ctggcgcgcc gctgttatac cctgcaagag gagaacaagc 1200 agctgcggcg tgcaggctgc ccctaccagg cagacgagaa ggtgaagcgg ctcaaggtaa 1260 agcgcgcgga gctgaccggg ctcgcgcggc gcctagctga ccgcgcccgc gagctgcagg 1320 agaccaacct ccgggccgtg agcgcgccta tacccggcga gagttgcgcc ggcctggagc 1380 tgtgccaagt ctttgcccgc cagcgcgctc gggacctgtc ggagcaggcg agcgcgccgc 1440 tggccaagga caagcagatc gaagagctgc ggcaggagtg ccacctcctg caggcgcgtg 1500 tcgcctcggg tccctgcagc gacctgcata ctggaagggg cggcccctgc acccagtggc 1560 tcaacgtcag agacttagac cgcctgcagc gcgagtccca gcgggaagtg ctgcgcctgc 1620 agaggcagtt gatgcttcag cagggcaacg gtggcgcttg gcccgaggcg ggcggccaga 1680 gcgcaacctg cgaggaggtg cgacggcaga tgctggcgct ggagcgcgag ctggaccagc 1740 ggcggcgcga gtgccaggag ctgggcgcgc aggcggcccc ggcgcggcga cgtggcgagg 1800 aggccgagac acagctgcag gcggcgctgc tcaaaaacgc ctggctggcg gaggagaatg 1860 ggcggctgca ggccaagacc gactgggtgc ggaaggtgga ggctgagaat agcgaagtgc 1920 gcggccacct gggccgcgcg tgtcaagagc gcgatgcctc cggcttgatc gccgaacagc 1980 tgctgcagca ggcggcgcgc gggcaggaca ggcagcagca gctgcaacgc gacccgcaga 2040 aggccctgtg tgacctccat ccttcctgga aggagataca ggcgctccag tgtcggcctg 2100 gtcaccctcc tgaacagccc tgggagacca gtcaaatgcc ggagtcccaa gttaaaggta 2160 gcagaaggcc caagttccac gcacggcctg aagactacgc agtgtcacag cccaacagag 2220 acatacagga gaaaagggaa gcctccctcg aggagagccc agttgccctt ggggagtcag 2280 ccagtgtccc ccaagtttca gagacagtcc ctgccagcca acctctgtcc aagaaaacca 2340 gctcccagtc aaactcctcc tctgaggggt cgatgtgggc caccgtgccg tcctccccta 2400 ctctggacag ggacacagcc agtgaggtgg atgacctgga gcctgacagc gtgtccctgg 2460 ccctggaaat ggggggctcg gcggctcctg ctgcccccaa gctcaagatc ttcatggctc 2520 agtataacta caacccattt gaggggccca atgatcaccc tgagggtgag ctgcccctca 2580 cagctgggga ctacatatat atcttcgggg acatggatga ggatggcttc tatgaggggg 2640 agcttgacga tggccggcgg gggctggtgc cctccaactt cgtggagcag attccggaca 2700 gctacatccc aggctgcctg cctgccaaat cccctgatct tggccccagt caactcccag 2760 cggggcagga tgaagctctg gaggaagaca gcttattatc tgggaaagcc cagggaatgg 2820 tggacagagg gctgtgccag atggtcaggg tgggctccaa gacagaagta gcaacagaga 2880 tcctggatac caagacggaa gcctgccagc tgggcttgct gcagagcatg gggaagcagg 2940 gcctctccag accccttctg gggaccaaag gggtgctccg tatggctccc atgcagctac 3000 acctgcagaa tgtcacagcc acatcagcca acatcacctg ggtctacagc agccaccgcc 3060 acccccatgt ggtatatctt gatgaccgag agcatgccct gaccccagcg ggcgtgagct 3120 gctacacctt ccagggcctg tgccccggca cgcactaccg ggtgcgggtg gaggtgcggc 3180 tgccatggga cttgctgcag gtgtattggg gaactatgtc ctccaccgtc accttcgaca 3240 cactcttggc aggacctccc tacccaccgc tggatgtgct ggtggagcgc catgcctcgc 3300 caggtgtcct ggtggtcagc tggctccctg tgaccattga ctcagctggg tcctccaatg 3360 gagtccaggt caccggttat gctgtgtatg cagatgggct taaggtttgt gaggtcgccg 3420 atgccactgc tgggagcacc gtattggaat tctcccagct acaggtgccc ctcacgtggc 3480 agaaggtctc agtgagaacc atgtcactct gtggtgagtc cctggattca gtgcctgctc 3540 agatccccga ggacttcttc atgtgtcacc gatggccaga gactccaccc tttagctaca 3600 cttgtggcga cccatccacc tacagagtca ccttccccgt ctgcccccag aagctgtcac 3660 tggctcctcc gagtgccaag gccagccccc acaaccctgg aagctgcggg gagccccagg 3720 ccaagttcct agaagcattc tttgaagaac ccccaaggag gcaatcccca gtgtccaacc 3780 tgggctcaga aggagaatgt ccgagttcag gggctggcag ccaagcccag gagcttgcag 3840 aggcctggga gggctgtaga aaggacctgc tctttcagaa gagtccccag aaccacaggc 3900 caccttcagt cagtgaccag cctggggaga aggaaaattg ctaccagcac atgggcacca 3960 gcaaaagccc tgctccagga ttcatccatc tacgcaccga gtgtgggccc aggaaagaac 4020 cgtgtcagga aaaggctgcc cttgagaggg tacttcggca aaagcaagat gcccaagggt 4080 tcacacctcc ccagctgggc gccagccaac agtatgcatc tgacttccat aacgttttga 4140 aggaggagca ggaggcactg tgcttggatc tgtggggcac agagaggcga gaggagagga 4200 gggagcctga gccccacagc aggcaaggac aagctctggg ggtcaagaga gggtgccagc 4260 tccatgagcc cagctcggca ctgtgtccag ctccatccgc caaagtcatc aagatgccca 4320 ggggtggccc ccaacagctg gggacggggg ccaacactcc agccagggtc tttgtggccc 4380 tctctgatta caaccccctg gtgatgtctg ccaacctcaa ggctgcagag gaggagctgg 4440 tcttccagaa aaggcagttg ctaagagtgt ggggctctca ggacacccat gatttctacc 4500 tcagcgagtg caacaggcaa gtgggcaata tccccgggcg cctagtggct gagatggagg 4560 tggggacaga gcagactgat aggaggtggc gttctccggc ccaagggcac ctgccttctg 4620 tggcccacct cgaggacttt caggggctca ccatccccca gggttcctcc ctggtgctcc 4680 aggggaactc caagagactc ccactgtgga ctccaaagat catgatagca gctctggact 4740 atgatcctgg ggatgggcaa atggggggcc aggggaaggg caggctggcg ctgagggcag 4800 gagacgtggt catggtttac gggcccatgg atgaccaagg attctattat ggagagttgg 4860 gcggccacag gggcctggtt cctgcccacc tgctggatca catgtccctc catggacact 4920 gagcaagcat ccttgcccag gtagtggcct ctggctgctc acaccctgcc agaggagaag 4980 caagcgttca gaccctcaca ccagcacccc tcctcaccac cataagtagc atgtgctcca 5040 agtgccactg tgttaaactg atggtagtcc ttaagcgtcc cctaggctct gaaagtagca 5100 ggacttaagc ctgagttatt tgcaaaagca aacacaacaa gccaacccct gagagtctga 5160 gaagccattt caaagttgct gataactatg gcaggtatac ggagaagcgc ctttttctgt 5220 ggccaatgtg tgttttctct gggaggttaa ggttatctgt ccattgcctt gtacgaaagt 5280 ctcaagaaaa gtctacatct taaaaaagaa aaagcaatct gagtgttatt tttgggatgt 5340 gagggtgatc tggctgcgac atgtgtcacc ccattgatca tcagggttga ttcggctgat 5400 ctggctgact aggcgggtat ccccttcctc cctcaccact ccatgtgcgt ccctccagaa 5460 gctgtgtgct caatggaaga ggatgaccat ccccgataga ggacgatcgg tcttcagtca 5520 agagtataag agtagctgcg ctcccctgct agaacctcca aacgagctct cagaatgtta 5580 tttttctgtc ctatgtccaa cccctcatta aaatgttcat agaaaaaa 5628 16 1482 DNA Homo sapiens misc_feature Incyte ID No 7476740CB1 16 tgggagacag cccccagaca gatgagtgtc gcgcctctct gagaggtgaa tgagcccgga 60 cggtccctac ctaccaagtc ctgaggagca gcggcaccaa cgacgcaggc ccgccccagc 120 ccgccagtga gccgcccatg ccctctgcta gcccggcccg cccgggcccc cgccatgctg 180 atcaccgtgt actgcgtgcg gagggacctc tccgaggtca ccttctctct ccaggtcagc 240 cccgactttg agctccgaaa cttcaaggtc ctctgcgaag cggagtccag agtccccgtc 300 gaagagatcc agatcatcca catggagcga ctcctcatcg aggaccactg ttccctgggc 360 tcctacggcc tcaaagatgg cgatatcgtg gttttactgc agaaggacaa tgtgggacct 420 cgggctccag ggcgtgcccc gaaccagcct cgtgtagact tcagtggcat tgcggtgcct 480 gggacgtcca gctcccgtcc acagcaccct ggacagcagc agcagcgcac acccgctgcc 540 cagcggtcac agggcttggc gtcaggagag aaggtggccg gcctgcaagg tctgggcagc 600 cccgccctga tccgcagcat gctgctctcc aacccccacg atctgtccct gctcaaggaa 660 cgcaaccctc ccttggcgga agccctgctc agcggaagcc ttgagacctt ttctcaggtg 720 ctgatggagc agcaaaggga aaaggccttg agagagcaag agaggcttcg tctctacaca 780 gccgacccac tggatcggga agctcaggcc aaaatagaag aggaaatccg gcagcaaaac 840 attgaagaaa acatgaatat agcgatagaa gaggcccccg agagttttgg acaagtgacg 900 atgctctaca ttaactgcaa agtgaatggg catcctttga aggcttttgt tgactcgggc 960 gcccagatga ccattatgag ccaggcttgt gccgagcgat gtaacatcat gaggctggtg 1020 gaccgacggt gggctggggt tgctaaagga gtgggcacac agagaattat tggccgtgtt 1080 catctagctc agattcaaat tgaaggtgat ttcttacagt gctctttctc catacttgag 1140 gatcaaccca tggatatgct tctaggccta gatatgctcc ggagacatca atgttccatc 1200 gatttgaaga aaaatgtgct ggtcatcggc accactggca cgcagactta ttttcttcct 1260 gagggagagt tgcccttatg ctctaggatg gtaagtgggc aagatgagtc ttcggacaag 1320 gaaattacac attcagtcat ggattcagga cgaaaagagc attaaagcac gttataaata 1380 tgttaccacc ttgagggagc ctcaggtccc cggcaattat aagttaagag cttactggca 1440 atgtaatcat taaaaaacat cagtaacaac taaaaaaaaa aa 1482 17 2511 DNA Homo sapiens misc_feature Incyte ID No 7473774CB1 17 gatgtgactg ttaagctgag ctttttctcc cggcctcagc ccctagatca gacattctct 60 ctcattaccc ggcgcgtggg aacgggtcca cagccccttg tccgccctag aacccccatg 120 ggctgccgcc cgccgccgcc tcggctgcca cccaggacac ggcagagata agcgcaggac 180 cagacggcca ccatgtcagg agactacgag gatgacctct gccggcgggc actcatcctg 240 gtctcggacc tctgtgcgcg ggtccgagat gctgacacca acgacaggtg ccaggagttc 300 aatgaccgaa tccgaggcta tccccggggt ccagatgcag acatctccgt gagcctgctg 360 tcggtcatcg tgacattctg tggcattgtc cttctgggtg tctctctctt cgtgtcctgg 420 aagttgtgct gggtgccctg gcgggacaag ggaggctcgg cagtgggcgg tggccccctg 480 cgcaaagacc taggccctgg tgtcgggctg gcaggcctgg taggcggagg cgggcaccac 540 ctggcggctg gcctgggtgg ccatcctctg ctgggcggcc cacaccacca tgcccatgcc 600 gcccaccatc caccctttgc tgagctgctg gagccaggca gcctgggggg ttctgacacc 660 cctgagccct cctacttgga catggactcg tatccagagg ctgcagcagc agcagtggcc 720 gctggggtca aaccgagcca aacatcccct gagctgccct ctgagggggg agcaggctct 780 gggttgctcc tgctgccccc cagtggtggg ggcttgccca gtgcccagtc acatcagcag 840 gtcacaagcc tggcacccac taccaggtac ccagccctgc cccgacccct cacccagcag 900 actctgacct cccagccgga ccccagcagt gaggagcgcc cacctgccct gcccttaccc 960 ctgcctggag gcgaggaaaa agccaaactc attgggcaga ttaagccaga gctgtaccag 1020 gggactggcc ctggtggccg gcggagcggt gggggcccag gctctggaga ggcaggcaca 1080 ggggcaccct gtggccgtat cagcttcgcc ctgcggtacc tctatggctc ggaccagctg 1140 gtggtgagga tcctgcaggc cctggacctc cctgccaagg actccaacgg cttctcagac 1200 ccctacgtca agatctacct gctgcctgac cgcaagaaaa agtttcagac caaggtgcac 1260 aggaagaccc tgaaccccgt cttcaatgag acgtttcaat tctcggtgcc cctggccgag 1320 ctggcccaac gcaaactgca cttcagcgtc tatgactttg accgcttctc gcggcacgac 1380 ctcatcggcc aggtggtgct ggacaacctc ctggagctgg ccgagcagcc ccctgaccgc 1440 ccgctctgga gggacatcgt ggagggcggc tcggaaaaag cagatcttgg ggagctcaac 1500 ttctcactct gctacctccc cacggccggg cgcctcaccg tgaccatcat caaagcctct 1560 aacctcaaag cgatggacct cactggcttc tcagacccct acgtgaaggc ctccctgatc 1620 agcgaggggc ggcgtctgaa gaagcggaaa acctccatca agaagaacac gctgaacccc 1680 acctataatg aggcgctggt gttcgacgtg gcccccgaga gcgtggagaa cgtggggctc 1740 agcatcgccg tggtggacta cgactgcatc gggcacaacg aggtgatcgg cgtgtgccgt 1800 gtgggccccg acgctgccga cccgcacggc cgcgagcact gggcagagat gctggccaat 1860 ccccgcaagc ccgtggagca ctggcatcag ctagtggagg aaaagactgt gaccagcttc 1920 acaaaaggca gcaaaggact atcagagaaa gagaactccg agtgaggggt ctggcctagg 1980 cccgggatcg gaccaggctc cctcaggacc ccatcctttc ctgcccggac cgtgaattca 2040 tctccttgaa gccataacgt ccgagctgct ggtgcggggc agccctggcc ctaggcttcc 2100 taaccctgga agcgagagga tgagaggagg ccggcccagc tccttctttc agggtggggg 2160 tcattcagcc tccactgtgt ctgtcttttc ttccctgggg ctccccctcg aggcgagggg 2220 ccatgcatgt ctgggggacc cctgcccccc aaaaccctct gtctgtctct gtctctttgc 2280 tgtttgtcca agactcagtg tcccgaccct tgttctcgcc gtgaatgtca atgggccaat 2340 cctctctgtc ctttcagaca cacacacacc tgtgtccacc ccttctgttc gccacaccct 2400 gcgtctggcc ggtcccccca ctgctgctgc tatcaacgcc agaataaaca cactctgtgg 2460 gtctcactcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 2511 18 1680 DNA Homo sapiens misc_feature Incyte ID No 7946329CB1 18 cctacctctc atcaggacca gtctgactgc acctgcatcc ttagctcaga gcatccccgg 60 agcatcttaa gagctgagcg cagctgacaa ctaggggccg gaccgtcgca ggaggcgtcc 120 gctggatacc ttcccccttc cctgacctag agctctacag ctgctgcctc ggtactgacc 180 gagggttccc agagctgtct taccattgca aaaacgttat agcaacagcc tctgattacg 240 acatggctga gatcaccaat atccgaccta gctttgatgt gtcaccggtg gtggccggcc 300 tcatcggggc ctctgtgctg gtggtgtgtg tctcggtgac cgtctttgtc tggtcatgct 360 gccaccagca ggcagagaag aagcacaaga acccaccata caagtttatt cacatgctca 420 aaggcatcag catataccca gagaccctca gcaacaagaa gaaaatcatc aaagtgcgga 480 gagacaaaga tggtcctggg agggaaggtg gacgtaggaa cctgttggtg gacgcagcag 540 aggctggcct gctaagccga gacaaagatc ccagggggcc tagctctgga tcttgtatag 600 accaattacc catcaaaatg gactatgggg aagaactaag gagccctatt acaagcctga 660 cccctgggga gagcaaaacc acctctccat catctccaga ggaggatgtc atgctaggat 720 ccctcacctt ctcagtggac tataacttcc cgaaaaaagc cctggtggtg acaatccagg 780 aggcccacgg gctgccagtg atggatgacc agacccaggg atctgacccc tacatcaaaa 840 tgaccatcct tcctgacaaa cggcatcggg tgaagaccag agtgctgcgg aagaccctgg 900 accctgtgtt tgacgagacc ttcaccttct atggcatccc ctacagccag ctgcaggacc 960 tggtgctgca cttccttgtc ctcagctttg accgcttctc tcgggatgat gtcattggcg 1020 aggtcatggt gccactggca ggggtggacc ccagcacagg caaggtacaa ctgaccaggg 1080 acatcatcaa aaggaatatc cagaagtgca tcagcagagg ggagctccag gtgtctctgt 1140 catatcagcc tgtggcacag agaatgacag tggtggtcct caaagccaga cacttgccga 1200 agatggatat caccggtctc tcaggtaatc cttatgtcaa ggtgaacgtc tactacggca 1260 gaaagcgcat tgccaagaag aaaacccatg tgaagaagtg cactttgaac cccatcttca 1320 atgaatcttt catctacgac atccccactg acctcctgcc tgatatcagc atcgagttcc 1380 tcgttatcga cttcgatcgc accaccaaga atgaggtggt ggggaggctg atcctggggg 1440 cacacagtgt cacagccagt ggtgctgaac actggagaga ggtctgcgag agcccccgca 1500 agcctgtggc caagtggcac agtctgagcg agtactaatc ctgttcttct ctcctctaat 1560 ccccgggggc caagctgggg agggatgtgg aggggaaaaa gatgacagag aagtggactc 1620 caaacctcat tttagttgta gaagaaaatt tcttacaaaa caaattccac aaagaacacc 1680 

What is claimed is:
 1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.
 2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO:1-9.
 3. An isolated polynucleotide encoding a polypeptide of claim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID NO:10-18.
 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 3. 7. A cell transformed with a recombinant polynucleotide of claim
 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim
 6. 9. A method for producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
 10. An isolated antibody which specifically binds to a polypeptide of claim
 1. 11. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:10-18, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).
 12. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim
 11. 13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 14. A method of claim 13, wherein the probe comprises at least 60 contiguous nucleotides.
 15. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 16. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
 17. A composition of claim 16, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.
 18. A method for treating a disease or condition associated with decreased expression of functional SAT, comprising administering to a patient in need of such treatment the composition of claim
 16. 19. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
 20. A composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.
 21. A method for treating a disease or condition associated with decreased expression of functional SAT, comprising administering to a patient in need of such treatment a composition of claim
 20. 22. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
 23. A composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.
 24. A method for treating a disease or condition associated with overexpression of functional SAT, comprising administering to a patient in need of such treatment a composition of claim
 23. 25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 1. 26. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, said method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim
 1. 27. A method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 28. A method for assessing toxicity of a test compound, said method comprising: a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 11 or fragment thereof; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 29. A diagnostic test for a condition or disease associated with the expression of SAT in a biological sample comprising the steps of: a) combining the biological sample with an antibody of claim 10, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex; and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
 30. The antibody of claim 10, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 31. A composition comprising an antibody of claim 10 and an acceptable excipient.
 32. A method of diagnosing a condition or disease associated with the expression of SAT in a subject, comprising administering to said subject an effective amount of the composition of claim
 31. 33. A composition of claim 31, wherein the antibody is labeled.
 34. A method of diagnosing a condition or disease associated with the expression of SAT in a subject, comprising administering to said subject an effective amount of the composition of claim
 33. 35. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 10 comprising: a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, or an immunogenic fragment thereof, under conditions to elicit an antibody response; b) isolating antibodies from said animal; and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.
 36. An antibody produced by a method of claim
 35. 37. A composition comprising the antibody of claim 36 and a suitable carrier.
 38. A method of making a monoclonal antibody with the specificity of the antibody of claim 10 comprising: a) immunizing an animal with a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9, or an immunogenic fragment thereof, under conditions to elicit an antibody response; b) isolating antibody producing cells from the animal; c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells; d) culturing the hybridoma cells; and e) isolating from the culture monoclonal antibody which binds specifically to a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.
 39. A monoclonal antibody produced by a method of claim
 38. 40. A composition comprising the antibody of claim 39 and a suitable carrier.
 41. The antibody of claim 10, wherein the antibody is produced by screening a Fab expression library.
 42. The antibody of claim 10, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 43. A method for detecting a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9 in a sample, comprising the steps of: a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9 in the sample.
 44. A method of purifying a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9 from a sample, the method comprising: a) incubating the antibody of claim 10 with a sample under conditions to allow specific binding of the antibody and the polypeptide; and b) separating the antibody from the sample and obtaining the purified polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-9.
 45. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:1.
 46. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:2.
 47. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:3.
 48. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:4.
 49. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:5.
 50. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:6.
 51. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID) NO:7.
 52. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:8.
 53. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:9.
 54. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:10.
 55. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID) NO:11.
 56. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:12.
 57. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:13.
 58. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:14.
 59. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:15.
 60. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:16.
 61. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:17.
 62. A polynucleotide of claim 11, comprising the polynucleotide sequence of SEQ ID NO:18. 